Anti-fungals compounds targeting the synthesis of fungal sphingolipids

ABSTRACT

The present invention provides a compound having the structure: 
                         
and use of the compound for inhibiting the growth of or killing a fungus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a § 371 national stage of PCT InternationalApplication No. PCT/US2018/037846, filed Jun. 15, 2018, claiming thebenefit of U.S. Provisional Application No. 62/521,069, filed Jun. 16,2017, the contents of each of which are hereby incorporated byreference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI116420 and AI100631 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

Throughout this application, certain publications are referenced inparentheses. Full citations for these publications may be foundimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention relates.

BACKGROUND OF THE INVENTION

Globally, over 300 million people are afflicted by a serious fungalinfection and 25 million are at risk of dying or losing their sight(Fungal Infection Trust 2011). Among fungal infections, invasive fungalinfections such as cryptococcosis, candidiasis, aspergillosis andpneumocystosis are the most common and the most life-threatening (Brown,G. D. et al. 2012; Gullo, A. 2009; Tuite, N. L. & Lacey. K. 2013). Theseinfections have risen dramatically over the last 20 years, some over14-fold. The CDC estimates that more than 1 million new cases per yearof cryptococcosis will occur worldwide in patients with AIDS, and600,000 will die from the infection. This is a drastic increaseconsidering that prior to the mid-1950s, fewer than 300 cases had beenreported in the medical literature (reviewed in Sorrell, T. C. et al.2011). Certain medical devices, such as catheters, provide the port ofentry to fungi that colonize the skin and mucosa. As a result,disseminated candidiasis is the 4th most common hospital-acquired sepsiswith >120,000 deaths/year (Perlroth, J. et al. 2007; Rueping, M. J. etal. 2009; Guery B. P. et al. 2009). Disseminated aspergillosisrepresents another invasive fungal infection that is steadily increasingin immunocompromised patients with a mortality rate of 450,000/year(Mayr, A. & Lass-Florl, C. 2011; Maschmeyer, G. et al. 2007; Munoz, P.et al. 2008; Ruping, M. J. et al. 2008). Aspergillus spp. is alsoresponsible for severe asthma by fungal sensitization (SAFS) accountingfor 100,000 additional deaths annually.

Pneumocystis spp. are a group of host-specific opportunistic fungi thatreside in the lungs of humans and animals in nature. The organism isnamed P. jirovecii in humans, P. carinii in rats, and P. murina in mice.Pneumocystis pneumonia (PCP) remains the most prevalent opportunisticinfection in patients infected with the human immunodeficiency virus(HIV). An estimated 539 million patients were discharged from hospitalsbetween 1986 and 2005, of whom an estimated 312,411 had AIDS-associatedPCP. Although numbers of cases of PCP has decreased in economicallydeveloped countries, the worldwide incidence is estimated to exceed400,000 (Kelley, C. F. et al. 2009). Reports on mortality rates for PCPare variable, ranging from 13% to as high as 80%, which even at thelowest rate results in more than 52,000 deaths per year (Kelley, C. F.et al. 2009). PCP is also prevalent in other patient groups, notablypatients that are chronically immune suppressed due to solid organtransplantation or due to chemotherapy for cancer or autoimmune disease.P. jirovecii (Pj) is also a frequent colonizer of the respiratory tractin immunocompetent individuals with other underlying pulmonary diseases,such as Chronic Obstructive Pulmonary Disease (COPD), in which itinitiates a deleterious inflammatory reaction (Huang, L. et al. 2006).Based on these reports, over 1,300,000 people are estimated to die everyyear because of invasive fungal infections and, most likely, this is anunderestimated figure (1, 2). This mortality rate is similar to the onefrom malaria (1,240,000/year) (WHO World Malaria Report 2013) andtuberculosis (1,400,000/year) (WHO World Global Tuberculosis 2013).

While there are about 30 branded prescription antifungal drugs on themarket, three classes of antifungals are mainly used to manage invasivefungal infections: 1) Azoles, such as fluconazole launched in themid-1980s, 2) polyenes, such as amphotericin B launched in the mid-1950sand 3) echinocandins, such as caspofungin launched in early 2000.However, the increased use of current azoles has led to an increase indrug resistance, limiting their effectiveness. In addition, drug-druginteraction issues can be a major impediment to the use of voriconazole,itraconazole and posaconazole. The interactions with cancer chemotherapyagents and immunosuppressants can be particularly difficult to handleclinically. Systemic antifungals, such as amphotericin B, tend to haverelatively high toxicity and side effects. The echinocandins have alower incidence of adverse events compared to older antifungals but theybind highly to serum proteins, there are no oral formulations, and theirantifungal spectrum of activity is very narrow (Farowski, F. et al.2012; Farowski, F. et al. 2013; Odabasi, Z. et al. 2007; Saribas, Z. etal. 2012; Yanni, S. B. et al. 2011). In the case of Pneumocystis, thesituation is direr. Pneumocystis pneumonia does not respond to any ofthe standard antifungals described above (Carmona, E. M. & Limper, A. H.2011). The drug of choice for the treatment and chemoprophylaxis of PCPis trimethoprim-sulfamethoxazoe (TMP-SMX). Analysis of P. jiroveciiisolates demonstrates that the pathogen is evolving mutations in thetarget genes of TMP-SMX, suggesting P. jirovecii could soon becomeresistant to SMX in the combination, considered the more potent of thetwo drugs that makeup the combination therapy (Ma, L. et al. 1999).Atovaquone and pentamidine, both second line treatments, suffer from lowefficacy and severe adverse events (SAEs) that include nephrotoxicity,neutropenia, hypotension and hypoglycemia (Benfield, T. et al. 2008).Atovaquone inhibits the mitochondrial cytochrome Bc1 complex inparasites at much lower concentrations than the respective mammaliancomplex. However, evolving resistance to atovaquone, corresponding tomutations in the Pneumocystis cytochrome b gene, has been observed(Kazanjian, P. et al. 2001). Pentamidine has a broad antimicrobialaction with no specific target known and is highly toxic and oftenconsidered to be a drug of last resort. We are faced, then, with agrowing patient population, a microorganism that cannot be easilysubjected to detailed biochemical analysis in the laboratory, adeveloping resistance to standard of care medications and a limitedindustrial effort to advance new therapies into the clinic. Thus, thereis a need for new, safer and more effective compounds.

Studies in our and other laboratories identified sphingolipids as keyregulators of fungal pathogenesis (reviewed in Heung, L. J. et al. 2006and Singh, A. 2011). Particularly, a fungal sphingolipid namedglucosylceramide (GlcCer) is required for the pathogenic fungusCryptococcus neoformans to cause a lethal meningo-encephalitis(Kechichian, T. B. et al. 2007; Rittershaus, P. C. et al. 2006). Infact, mice survived the infection by a C. neoformans mutant strainlacking the final enzyme for the synthesis of GlcCer (GlcCer synthase 1or Gcs1). The Δgcs1 mutant was confined in the lung granuloma and it didnot reach the bloodstream and, thus, it did not disseminate to thebrain. Later, other investigators corroborated and extended our findingsthat mutation of genes involved in the last steps of the GlcCer pathwayaffect fungal virulence not only of fungi infecting humans, such as C.neoformans (Liu, O. W. et al. 2008; Singh, A. et al. 2012), Candidaalbicans (Oura, T. & Kajiwara, S. 2010; Noble, S. M. et al. 2010; Oura,T. & Kajiwara, S. 2008), and Aspergillus fumigatus (Levery, S. B. et al.2002), but also of fungi infecting plants (da Silva, A. F. et al. 2004;Ramaoorthy, V. et al. 2009). That GlcCer is required for fungalvirulence in plants is also suggested by studies showing that plantsdefend themselves against fungi by producing specific defensins (e.g.RsAFP2 and others) that bind to fungal and not mammalian GlcCer(Thevissen, K. et al. 2004). Interestingly, these plant defensins areable to bind GlcCer of human pathogenic fungi and able to kill them invitro and, in some cases, during in vivo infection in animal models(Thevissen, K. 2004; Tavares, P M. 2008; Aerts, A M. 2007; Lobo, D S.2007; Thevissen, K. 2012; Mello Ede, O. et al. 2014; Oguro, Y. et al.2014; Goncalves, S. et al. 2012; de Medeiros, L. N. et al. 2010).

Mechanistic studies revealed that GlcCer is involved in the regulationof fungal cell replication in environments characterized byneutral/alkaline pH (Kechichian, T. B. et al. 2007; Levery, S. B. et al.2002; Rhome, R. et al. 2011). Particularly, when fungal cells lackingGlcCer are exposed to neutral/alkaline pH, they cannot progress throughthe cell cycle and, thus, cytokinesis does not occur (Rittershaus, P. C.et al. 2006; Levery, S. B. et al. 2002; Saito, K. et al. 2006). Later,we linked this phenomenon to the regulation by GlcCer of physicalproperties of fungal plasma membranes of C. neoformans (Singh, A. et al.2012). The synthesis of GlcCer seems to be important also duringPneumocystis pneumonia (PCP) as GlcCer synthase transcripts have beenfound to be elevated at the time of isolation of the fungus from afulminate lung infection (Cushion, M. T. et al. 2007). Interestingly, inmost dimorphic fungi, production of GlcCer is detected only in the hostinfective form (yeast) and not in the environmental form (mold)(Warnecke, D. et al. 2003; Rhome, R. et al. 2007; Toledo, M. S. et al.2001). Taken together, these studies suggest that GlcCer is most likelya pan-fungal virulence factor required during infection to promotefungal growth at neutral/alkaline environments in the host (e.g.alveolar spaces, cerebrospinal fluid and bloodstream), and as such, itis a promising novel drug target. Currently, inhibitors that block thefungal but not the mammalian GlcCer synthesis are not available.

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, or    -   R₃ and R₄ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₂, —OCHF₂ or —OCF₃, and R₅ and R₆        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted, or    -   R₃ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substitute, or    -   R₅ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted; and    -   A is an aryl or heteroaryl, which are each unsubstituted or        substituted,    -   wherein when R₃, R₄, and R₆ are each —H and R₅ is —OH or —OCH₃,        or R₃, R₅, and R₆ are each —H and R₄ is —Br, then A is other        than ortho-tolyl or meta-bromophenyl,    -   or a pharmaceutically acceptable salt or ester thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Compound 1 killed C. neoformans in a concentration dependentmanner.

FIG. 1B. Compound 2 killed C. neoformans in a time dependent manner.

FIG. 2. In vivo efficacy evaluation (survival study) of compound A15 inan animal model.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, or    -   R₃ and R₄ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted, or    -   R₃ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substitute, or    -   R₅ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted; and    -   A is an aryl or heteroaryl, which are each unsubstituted or        substituted,    -   wherein when R₃, R₄, and R₆ are each —H and R₅ is —OH or —OCH₃,        or R₃, R₅, and R₆ are each —H and R₄ is —Br, then A is other        than ortho-tolyl or meta-bromophenyl,    -   or a pharmaceutically acceptable salt or ester thereof.

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H;    -   R₂ is —H;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, or    -   R₃ and R₄ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted, or    -   R₃ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substitute, or    -   R₅ and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH,        —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄        combine to form a fused aryl or fused heteroaryl, which are each        unsubstituted or substituted; and    -   A is a phenyl, which is unsubstituted or substituted,    -   wherein when R₃, R₄, and R₆ are each —H and R₅ is —OH or —OCH₃,        or R₃, R₅, and R₆ are each —H and R₄ is —Br, then A is other        than ortho-tolyl or meta-bromophenyl,    -   or a pharmaceutically acceptable salt or ester thereof.

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H;    -   R₂ is —H;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; and    -   A is a phenyl, which is unsubstituted or substituted,    -   wherein when R₃, R₄, and R₆ are each —H and R₅ is —OH or —OCH₃,        or R₃, R₅, and R₆ are each —H and R₄ is —Br, then A is other        than ortho-tolyl or meta-bromophenyl,    -   or a pharmaceutically acceptable salt or ester thereof.

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H;    -   R₂ is —H;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; and    -   A is a phenyl, which disubstituted,    -   or a pharmaceutically acceptable salt or ester thereof.

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H;    -   R₂ is —H;    -   R₃, R₄, R₅, and R₆ are each independently —H, halogen, C₁-C₆        alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; and    -   A is a phenyl, which trisubstituted,    -   or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, wherein when R₃ and R₅ are each —H and R₄ and R₆are each —Br, then A is other than para-bromophenyl, meta-bromophenyl,ortho-tolyl or 3-quinolinyl.

In some embodiments, wherein when R₃, R₅ and R₆ are each —H and R₄ is—Br, then A is other than 3,5-dibromo-ortho-hydorxyphenyl,para-bromophenyl, meta-bromophenyl or ortho-tolyl.

In some embodiments, wherein when R₃ and R₅ are each —H and R₄ and R₆are each —Br, then A is other than para-bromophenyl, meta-bromophenyl,ortho-tolyl or 3-quinolinyl; and when R₃, R₅ and R₆ are each —H and R₄is —Br, then A is other than 3,5-dibromo-ortho-hydorxy,para-bromophenyl, meta-bromophenyl or ortho-tolyl.

In some embodiments, wherein when R₃ and R₅ are each —H and R₄ and R₆are each —Br, then A is other than para-bromophenyl; and when R₃, R₅ andR₆ are each —H and R₄ is —Br, then A is other than3,5-dibromo-ortho-hydorxyphenyl.

In some embodiments, A is other than para-bromophenyl, meta-bromophenyl,ortho-tolyl or 3-quinolinyl.

In some embodiments, A is other than 3,5-dibromo-ortho-hydorxyphenyl,para-bromophenyl, meta-bromophenyl or ortho-tolyl

In some embodiments, A is other than ortho-tolyl or meta-bromophenyl.

In some embodiments, A is other than 3,5-dibromo-ortho-hydorxyphenyl,para-bromophenyl, meta-bromophenyl, ortho-tolyl or 3-quinolinyl.

In some embodiments, wherein the aryl or heteroaryl is substituted withhalogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃.

In some embodiments, wherein the fused aryl or fused heteroaryl issubstituted with halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃.

In some embodiments, the compound wherein one of R₃-R₆ is other than —H.

In some embodiments, the compound wherein two of R₃-R₆ is other than —H.

In some embodiments, the compound wherein A is monosubstituted.

In some embodiments, the compound wherein A is disubstituted.

In some embodiments, the compound wherein A is trisubstituted.

In some embodiments, the compound wherein R₃, R₄, R₅, and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃.

In some embodiments, the compound wherein R₃, R₄, R₅, and R₆ are eachindependently halogen, —O—(C₁-C₆ alkyl), —OCF₃ or —CF₃.

In some embodiments, the compound wherein R₃, R₄, R₆, and R₆ are eachindependently halogen or —O—(C₁-C₆ alkyl).

In some embodiments, the compound wherein R₃, R₄, R₅, and R₆ are eachindependently —Cl, —Br, —F, —O—(C₁-C₆ alkyl), —OCF₃ or —CF₃.

In some embodiments, the compound wherein R₃, R₄, R₅, and R₆ are eachindependently —Cl, —Br, or —O—(C₁-C₆ alkyl).

In some embodiments, the compound wherein

-   -   R₃ is —H, R₄ is —CH₃, Cl or Br, R₅ is —H, and R₆ is —CH₃, Cl or        Br; or    -   R₃ is —H, R₄ is —CH₃, Cl or Br, R₅ is —H, and R₆ is —H; or    -   R₃ is —H, R₄ is —H, R₅ is —CH₃, Cl or Br, and R₆ is —H.

In some embodiments, the compound having the structure:

In some embodiments, the compound wherein

-   -   R₃ is —H, R₄ is —F, —OCF₃ or —CF₃, R₅ is —H, and R₆ is —H; or    -   R₃ is —H, R₄ is —H, R₅ is —H, and R₆ is Cl or Br.

In some embodiments, the compound having the structure:

In some embodiments, the compound wherein R₃ and R₄ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆ combine to form a fused aryl orfused heteroaryl, which are each unsubstituted or substituted withhalogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃.

In some embodiments, the compound wherein R₃ and R₄ are eachindependently —H, halogen, C₁—C alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆ combine to form a fusedunsubstituted phenyl.

In some embodiments, the compound wherein R₃ and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅ combine to form a fused aryl orfused heteroaryl, which are each unsubstituted or substituted withhalogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃.

In some embodiments, the compound wherein R₃ and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂, or —OCF₃, and R₄ and R₅ combine to form a fusedunsubstituted phenyl.

In some embodiments, the compound wherein R₅ and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄ combine to form a fused aryl orfused heteroaryl, which are each unsubstituted or substituted withhalogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃.

In some embodiments, the compound wherein R₅ and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄ combine to form a fusedunsubstituted phenyl.

In some embodiments, the compound having the structure:

In some embodiments, the compound wherein A is an unsubstituted aryl.

In some embodiments, the compound wherein A is a substituted aryl.

In some embodiments, the compound wherein A is an unsubstitutedheteroaryl.

In some embodiments, the compound wherein A is a substituted heteroaryl.

In some embodiments, the compound wherein the aryl is a phenyl,l-naphthyl or 2-naphthyl.

In some embodiments, the compound wherein the heteroaryl is a pyridinyl,pyrrolyl, thienyl, furyl, quinolyl, isoquinolyl, indolyl, benzothienylor benzofuryl.

In some embodiments, the compound wherein A has the structure:

wherein R₇, R₈, R₉, R₁₀ and R₁₁ are each, independently, —H, halogen,C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂, —OCF₃, —CN,—CH₂OCH₃, —N(CH₃)₂, —CH₂F, —N₃ or —CCH.

In some embodiments, the compound wherein A has the structure:

wherein R₇, R₈, R₉, R₁₀ and R₁₁ are each, independently, —H, halogen,C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃.

In some embodiments, the compound wherein R₇, R₈, R₉, R₁₀ and R₁₁ areeach, independently, halogen or —O—(C₁-C₆ alkyl).

In some embodiments, the compound wherein R₇, R₈, R₉, R₁₀ and R₁₁ areeach, independently, —Cl, —Br, or —O—(C₁-C₆ alkyl).

In some embodiments, the compound wherein one of R₇ or R₁₁ is —H.

In some embodiments, the compound wherein A has the structure:

In some embodiments, the compound wherein A has the structure:

In some embodiments, the compound wherein A has the structure:

In some embodiments, the compound wherein A has the structure:

In some embodiments, the compound having the structure:

In some embodiments, the compound wherein R₁ is —H or —CH₃; and R₂ is —Hor —CH₃. In some embodiments, the compound wherein R₁ is —H; and R₂ is—H.

In some embodiments, the compound having the structure:

-   -   or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, the compound having the structure:

-   -   or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, the compound having the structure:

-   -   or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention and a pharmaceutically acceptablecarrier.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention, an anti-fungal agent and apharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition the anti-fungal agentis fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidinA.

The present invention provides a method of inhibiting the growth of afungus comprising contacting the fungus with an effective amount of thecompound of the present invention or a pharmaceutically acceptable saltor ester thereof, so as to thereby inhibit the growth of the fungus.

The present invention also provides a method of inhibiting fungalsphingolipid synthesis in a fungus comprising contacting the fungus withan effective amount of the compound of the present invention or apharmaceutically acceptable salt or ester thereof, so as to therebyinhibit sphingolipid synthesis in the fungus.

The present invention further provides a method of inhibiting fungalsphingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian sphingolipid synthesis comprising administering tothe mammal an effective amount of the compound of the present inventionor a pharmaceutically acceptable salt or ester thereof, so as to therebyinhibit fungal sphingolipid synthesis in the fungus in the mammalwithout substantially inhibiting mammalian sphingolipid synthesis.

In some embodiments of the method, the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments of the method, the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments of the method, the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments of the method, further comprising contacting thefungus with an effective amount of an anti-fungal agent.

In some embodiments of the method, further comprising administering tothe mammal an effective amount of an anti-fungal agent.

In some embodiments of the method, wherein the amount of the compoundand the amount of the anti-fungal agent when taken together is moreeffective to inhibit the growth of the fungus than the anti-fungal agentalone, or more effective to inhibit fungal sphingolipid synthesis thanthe anti-fungal agent alone.

In some embodiments of the method, wherein the amount of the compoundand the amount of the anti-fungal agent when taken together is moreeffective to inhibit fungal sphingolipid synthesis without substantiallyinhibiting mammalian sphingolipid synthesis in the mammal than theanti-fungal agent alone.

In some embodiments of the method, wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments of the method, wherein the fungus is CryptococcusNeoformans, Cryptococcus gattii, Candida albicans, Candida krusei,Candida glabrata, Candida parapsilosis, Candida guilliermondii,Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp., Blastomycesdermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomycesvariotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasmacapsulatum, Aspergillus spp., dimorphic fungi or mucorales fungi.

In some embodiments of the method, wherein the fungus is other thanCryptococcus Neoformans.

In some embodiments of the method, wherein the fungus is Cryptococcusgattii, Candida albicans, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopusoryzae, Rhizopus spp., Blastomyces dermatitis, Histoplasma capsulatum,Coccidioides spp., Paecilomyces variotii, Pneumocystis murina,Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp.,dimorphic fungi or mucorales fungi.

In some embodiments of the method, wherein the fungal sphingolipid isglucosylceramide (GlcCer).

The present invention yet further provides a method of inhibiting thegrowth of or killing a fungus in a subject or treating a subjectafflicted with a fungal infection comprising administering to thesubject an effective amount of the compound of the present invention, ora pharmaceutically acceptable salt or ester thereof, so as to therebyinhibiting the growth of or kill the fungus in the subject or treat thesubject afflicted with the fungal infection.

In some embodiments of the method, further comprising administering aneffective amount of an anti-fungal agent.

In some embodiments of the method, wherein the amount of the compoundand the amount of the anti-fungal agent when taken together is moreeffective to treat the subject than when the anti-fungal agent isadministered alone.

In some embodiments of the method, wherein the amount of the compoundand the amount of the anti-fungal agent when taken together is effectiveto reduce a clinical symptom of the fungal infection in the subject.

In some embodiments of the method, wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments of the method, wherein the fungal infection iscaused by Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis,Stachybotrys or Mycrorales fungus.

In some embodiments of the method, wherein the fungal infection iscaused by Cryptococcus Neoformans.

In some embodiments of the method, wherein the fungal infection isCryptococcus neoformans cryptococcosis.

In some embodiments of the method, wherein the fungal infection iscaused by a fungus other than Cryptococcus Neoformans.

In some embodiments of the method, wherein the fungal infection is afungal infection other than Cryptococcus neoformans cryptococcosis.

In some embodiments of the method, wherein the fungal infection isAspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,Cryptococcus gattii cryptococcosis, Fungal Keratitis, Dermatophytes,Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), orSporotrichosis.

In some embodiments of the method, wherein the fungal infection iscaused by Cryptococcus gattii, Candida albicans, Candida krusei, Candidaglabrata, Candida parapsilosis, Candida guilliermondii, Aspergillusfumigatus, Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis,Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii,Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum,Aspergillus spp., or dimorphic fungi.

The present invention yet further provides a compound having thefollowing structure:

or a pharmaceutically acceptable salt thereof, for use in inhibiting thegrowth of a fungus.

The present invention yet further provides a compound having thefollowing structure:

or a pharmaceutically acceptable salt thereof, for use in inhibitingfungal sphingolipid synthesis in a fungus.

The present invention yet further provides a compound having thefollowing structure:

or a pharmaceutically acceptable salt thereof, for use in inhibitingfungal sphingolipid synthesis in a fungus in a mammal.

The present invention yet further provides a compound having thefollowing structure:

or a pharmaceutically acceptable salt thereof, for use in inhibiting thegrowth of or killing a fungus in a subject or treating a subjectafflicted with a fungal infection caused by the fungus.

In some embodiments the compound for use wherein the fungus is otherthan Cryptococcus Neoformans.

In some embodiments the compound for use wherein the fungal infection iscaused by a fungus other than Cryptococcus Neoformans.

In some embodiments the compound for use wherein the fungal infection isa fungal infection other than Cryptococcus neoformans cryptococcosis.

In some embodiments the compound for use further comprising ananti-fungal agent.

In some embodiments the compound for use wherein the anti-fungal agentis fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidinA.

The present invention yet further provides a pharmaceutical compositioncomprising the compound having the following structure:

or a pharmaceutically acceptable salt thereof, an anti-fungal agent anda pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition wherein theanti-fungal agent is fluconazole, amphotericin B, caspofungin,tunicamycin or aureobasidin A.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, for use in inhibiting thegrowth of a fungus.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, for use in inhibitingfungal sphingolipid synthesis in a fungus.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, for use in inhibitingfungal sphingolipid synthesis in a fungus in a mammal.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, for use in inhibiting thegrowth of or killing a fungus in a subject or treating a subjectafflicted with a fungal infection caused by the fungus.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, and an anti-fungal agentfor use in inhibiting the growth of a fungus.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, and an anti-fungal agentfor use in inhibiting fungal sphingolipid synthesis in a fungus.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, and an anti-fungal agentfor use in inhibiting fungal sphingolipid synthesis in a fungus in amammal.

The present invention yet further provides a pharmaceutical compositioncomprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof, and an anti-fungal agentfor use in inhibiting the growth of or killing a fungus in a subject ortreating a subject afflicted with a fungal infection caused by thefungus.

In some embodiments, the method wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments, the method wherein the fungal infection is causedby Candida, Aspergilius, Cryptococcus, Histoplasma, Pneumocystis,Stachybotrys or Mycrorales fungus.

In some embodiments, the method wherein the fungal infection isAspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,Cryptococcus gattii cryptococcosis, Fungal Keratitis, Dermatophytes,Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), orSporotrichosis.

In some embodiments, the method wherein the fungal infection is causedby Cryptococcus gattii, Candida aibicans, Candida krusei, Candidagiabrata, Candida parapsilosis, Candida guilliermondii, Aspergillusfumigatus, Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis,Histoplasma capsulatum, Coccidioides spp., Paeciiomyces variotii,Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, ordimorphic fungi.

In some embodiments, the fungal infection is an internal fungalinfection.

In some embodiments, the fungal infection is an invasive fungalinfection.

In some embodiments, the fungal infection is a fungal infection of theskin or lung. In some embodiments, the compound has a fungistatic effecton the fungus. In some embodiments, the compound has a fungicidal effecton the fungus. In some embodiments, the compound is administered orallyto the subject. In some embodiments, the compound is administeredtopically to the subject. In some embodiments, the subject is alsoafflicted with an immunodeficiency disorder. In some embodiments, thesubject is also afflicted with human immunodeficiency virus (HIV).

In some embodiments, the antifungal agent is Amphotericin B, Candicidin,Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Clotrimazole,Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole,Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole,Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole,Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole,Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine,Terbinafine, Anidulafungin, Caspofungin, Micafungin, Ciclopirox,Flucytosine, Griseofulvin, Haloprogin, Tolnaftate, or Undecylenic acid.

In some embodiments, a pharmaceutical composition comprising a compoundof the present invention and an antifungal agent, and at least onepharmaceutically acceptable carrier for use in treating a fungalinfection.

In some embodiments, a pharmaceutical composition comprising an amountof the compound of the present invention for use in treating a subjectafflicted with a fungal infection as an add-on therapy or in combinationwith, or simultaneously, contemporaneously or concomitantly with ananti-fungal agent.

In some embodiments of any of the above methods or uses, the subject isa human. In some embodiments of any of the above methods or uses, thecompound and/or anti-fungal agent is orally administered to the subject.In some embodiments of any of the above methods or uses, the compoundand/or anti-fungal agent is topically administered to the subject.

In some embodiments, the fungus or fungal infection has developedresistance to one or more drugs. For example, a drug resistant fungalinfection may have developed drug-resistance to an azole antifungaldrug, a polyene antifungal drug and/or an echinocandin antifungal drug.

In some embodiments of any of the above methods or uses, the compoundtargets APL5, COS111, XMKK1, and STE2 in the fungus. In some embodimentsof any of the above methods or uses, the compound targets at least oneof APL5, COS111, MKK1, or STE2 in the fungus. In some embodiments of anyof the above methods or uses, the compound disrupts vesicular transportmediate by APL5. In some embodiments of any of the above methods oruses, the fungus carries non-mutated APL5, COS111, MKK1, and STE2. Insome embodiments of any of the above methods or uses, the fungus carriesat least one of non-mutated APL5, COS111, MKK1, and STE2.

As used herein, a “symptom” associated with a fungal infection includesany clinical or laboratory manifestation associated with the fungalinfection and is not limited to what the subject can feel or observe.

As used herein, “treating”, e.g. of a fungal infection, encompassesinducing prevention, inhibition, regression, or stasis of the disease ora symptom or condition associated with the infection.

Compound 1 (ID #5271226), Compound 2 (ID #5281029), Compound 13 (ID#5475098), and Compound 17 (ID #5275737), are available fromChemBridge™, San Diego, Calif.

The contents of International Application Publication No.WO/2016/094307, published Jun. 16, 2016, are hereby incorporated byreference.

The compounds of the present invention include all hydrates, solvates,and complexes of the compounds used by this invention. If a chiralcenter or another form of an isomeric center is present in a compound ofthe present invention, all forms of such isomer or isomers, includingenantiomers and diastereomers, are intended to be covered herein.Compounds containing a chiral center may be used as a racemic mixture,an enantiomerically enriched mixture, or the racemic mixture may beseparated using well-known techniques and an individual enantiomer maybe used alone. The compounds described in the present invention are inracemic form or as individual enantiomers. The enantiomers can beseparated using known techniques, such as those described in Pure andApplied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in whichcompounds have unsaturated carbon-carbon double bonds, both the cis (Z)and trans (E) isomers are within the scope of this invention.

The compounds of the subject invention may have spontaneous tautomericforms. In cases wherein compounds may exist in tautomeric forms, such asketo-enol tautomers, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form.

In the compound structures depicted herein, hydrogen atoms are not shownfor carbon atoms having less than four bonds to non-hydrogen atoms.However, it is understood that enough hydrogen atoms exist on saidcarbon atoms to satisfy the octet rule.

This invention also provides isotopic variants of the compoundsdisclosed herein, including wherein the isotopic atom is ²H and/orwherein the isotopic atom ¹³C. Accordingly, in the compounds providedherein hydrogen can be enriched in the deuterium isotope. It is to beunderstood that the invention encompasses all such isotopic forms.

It is understood that the structures described in the embodiments of themethods hereinabove can be the same as the structures of the compoundsdescribed hereinabove.

It is understood that where a numerical range is recited herein, thepresent invention contemplates each integer between, and including, theupper and lower limits, unless otherwise stated.

Except where otherwise specified, if the structure of a compound of thisinvention includes an asymmetric carbon atom, it is understood that thecompound occurs as a racemate, racemic mixture, and isolated singleenantiomer. All such isomeric forms of these compounds are expresslyincluded in this invention. Except where otherwise specified, eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolutionmay be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atomsoccurring on the compounds disclosed herein. Isotopes include thoseatoms having the same atomic number but different mass numbers. By wayof general example and without limitation, isotopes of hydrogen includetritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughoutthis application, when used without further notation, are intended torepresent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore,any compounds containing ¹³C or ¹⁴C may specifically have the structureof any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structuresthroughout this application, when used without further notation, areintended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H.Furthermore, any compounds containing ²H or ³H may specifically have thestructure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art using appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

In the compounds used in the method of the present invention, thesubstituents may be substituted or unsubstituted, unless specificallydefined otherwise.

In the compounds used in the method of the present invention, alkyl,heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groupscan be further substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl andaminocarbonyl and aminothiocarbonyl.

It is understood that substituents and substitution patterns on thecompounds used in the method of the present invention can be selected byone of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art from readily available starting materials. If asubstituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or ondifferent carbons, so long as a stable structure results.

In choosing the compounds used in the method of the present invention,one of ordinary skill in the art will recognize that the varioussubstituents, i.e. R₁, R₂, etc. are to be chosen in conformity withwell-known principles of chemical structure connectivity.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. Thus, C₁-C_(n) as in “C₁-C_(n) alkyl”is defined to include groups having 1, 2 . . . , n−1 or n carbons in alinear or branched arrangement, and specifically includes methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl andso on. An embodiment can be C₁-C₁₂ alkyl, C₂-C₁₂ alkyl, C₃-C₁₂ alkyl,C₄-C₁₂ alkyl and so on. “Alkoxy” represents an alkyl group as describedabove attached through an oxygen bridge.

The term “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present. Thus, C₂-C_(n) alkenyl isdefined to include groups having 1, 2 . . . , n−1 or n carbons. Forexample, “C₂-C₆ alkenyl” means an alkenyl radical having 2, 3, 4, 5, or6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, forexample, 3 carbon-carbon double bonds in the case of a C₆ alkenyl,respectively. Alkenyl groups include ethenyl, propenyl, butenyl andcyclohexenyl. As described above with respect to alkyl, the straight,branched or cyclic portion of the alkenyl group may contain double bondsand may be substituted if a substituted alkenyl group is indicated. Anembodiment can be C₂-C₁₂ alkenyl, C₃-C₁₂ alkenyl, C₄-C₁₂ alkenyl and soon.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present. Thus, C₂-C_(n) alkynyl is defined to include groups having1, 2 . . . , n−1 or n carbons. For example, “C₂-C₆ alkynyl” means analkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triplebond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triplebonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds.Alkynyl groups include ethynyl, propynyl and butynyl. As described abovewith respect to alkyl, the straight or branched portion of the alkynylgroup may contain triple bonds and may be substituted if a substitutedalkynyl group is indicated. An embodiment can be a C₂-C_(n) alkynyl. Anembodiment can be C₁-C₁₂ alkynyl, C₃-C₁₂ alkynyl, C₄-C₁₂ alkynyl and soon

“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, adivalent alkane, alkene and alkyne radical, respectively. It isunderstood that an alkylene, alkenylene, and alkynylene may be straightor branched. An alkylene, alkenylene, and alkynylene may beunsubstituted or substituted.

As used herein, “heteroalkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and at least 1 heteroatom within the chain or branch.

As used herein, “heterocycle” or “heterocyclyl” as used herein isintended to mean a 5- to 10-membered nonaromatic ring containing from 1to 4 heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, but is notlimited to the following: imidazolyl, piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl,dihydropiperidinyl, tetrahydrothiophenyl and the like. If theheterocycle contains a nitrogen, it is understood that the correspondingN-oxides thereof are also encompassed by this definition.

As herein, “cycloalkyl” shall mean cyclic rings of alkanes of three toeight total carbon atoms, or any number within this range (i.e.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl orcyclooctyl).

As used herein, “monocycle” includes any stable polyatomic carbon ringof up to 10 atoms and may be unsubstituted or substituted. Examples ofsuch non-aromatic monocycle elements include but are not limited to:cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of sucharomatic monocycle elements include but are not limited to: phenyl.

As used herein, “bicycle” includes any stable polyatomic carbon ring ofup to 10 atoms that is fused to a polyatomic carbon ring of up to 10atoms with each ring being independently unsubstituted or substituted.Examples of such non-aromatic bicycle elements include but are notlimited to: decahydronaphthalene. Examples of such aromatic bicycleelements include but are not limited to: naphthalene.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include phenyl, p-toluenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl,phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituentis bicyclic and one ring is non-aromatic, it is understood thatattachment is via the aromatic ring.

As used herein, the term “polycyclic” refers to unsaturated or partiallyunsaturated multiple fused ring structures, which may be unsubstitutedor substituted.

The term “arylalkyl” refers to alkyl groups as described above whereinone or more bonds to hydrogen contained therein are replaced by a bondto an aryl group as described above. It is understood that an“arylalkyl” group is connected to a core molecule through a bond fromthe alkyl group and that the aryl group acts as a substituent on thealkyl group. Examples of arylalkyl moieties include, but are not limitedto, benzyl (phenylmethyl), p-trifluoromethylbenzyl(4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl,3-phenylpropyl, 2-phenylpropyl and the like.

The term “heteroaryl”, as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms selectedfrom the group consisting of O, N and S. Bicyclic aromatic heteroarylgroups include phenyl, pyridine, pyrimidine or pyridizine rings that are(a) fused to a 6-membered aromatic (unsaturated) heterocyclic ringhaving one nitrogen atom; (b) fused to a 5- or 6-membered aromatic(unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused toa 5-membered aromatic (unsaturated) heterocyclic ring having onenitrogen atom together with either one oxygen or one sulfur atom; or (d)fused to a 5-membered aromatic (unsaturated) heterocyclic ring havingone heteroatom selected from O, N or S. Heteroaryl groups within thescope of this definition include but are not limited to:benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl,thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The term “alkylheteroaryl” refers to alkyl groups as described abovewherein one or more bonds to hydrogen contained therein are replaced bya bond to an heteroaryl group as described above. It is understood thatan “alkylheteroaryl” group is connected to a core molecule through abond from the alkyl group and that the heteroaryl group acts as asubstituent on the alkyl group. Examples of alkylheteroaryl moietiesinclude, but are not limited to, —CH₂—(C₅H₄N), —CH₂—CH₂—(C₅H₄N) and thelike.

The term “heterocycle” or “heterocyclyl” refers to a mono- orpoly-cyclic ring system which can be saturated or contains one or moredegrees of unsaturation and contains one or more heteroatoms. Preferredheteroatoms include N, O, and/or S, including N-oxides, sulfur oxides,and dioxides. Preferably the ring is three to ten-membered and is eithersaturated or has one or more degrees of unsaturation. The heterocyclemay be unsubstituted or substituted, with multiple degrees ofsubstitution being allowed. Such rings may be optionally fused to one ormore of another “heterocyclic” ring(s), heteroaryl ring(s), arylring(s), or cycloalkyl ring(s). Examples of heterocycles include, butare not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane,piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine,tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclylsubstituents may be substituted or unsubstituted, unless specificallydefined otherwise. In the compounds of the present invention, alkyl,alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can befurther substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

As used herein, the term “halogen” refers to F, Cl, Br, and I.

The terms “substitution”, “substituted” and “substituent” refer to afunctional group as described above in which one or more bonds to ahydrogen atom contained therein are replaced by a bond to non-hydrogenor non-carbon atoms, provided that normal valencies are maintained andthat the substitution results in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Examples of substituent groups includethe functional groups described above, and halogens (i.e., F, Cl, Br,and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl,n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, suchas methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such asphenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; amino groups, such as amino, methylamino,dimethylamino, ethylamino, and diethylamino; and carboxyl.

Where multiple substituent moieties are disclosed or claimed, thesubstituted compound can be independently substituted by one or more ofthe disclosed or claimed substituent moieties, singly or pluraly. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

The various R groups attached to the aromatic rings of the compoundsdisclosed herein may be added to the rings by standard procedures, forexample those set forth in Advanced Organic Chemistry: Part B: Reactionand Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed.Edition. (2007), the content of which is hereby incorporated byreference.

The compounds used in the method of the present invention may beprepared by techniques well known in organic synthesis and familiar to apractitioner ordinarily skilled in the art. However, these may not bethe only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may beprepared by techniques described in Vogel's Textbook of PracticalOrganic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J.Hannaford, P. W. G. Smith, (Prentice Hall) 5^(th) Edition (1996),March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5^(th)Edition (2007), and references therein, which are incorporated byreference herein. However, these may not be the only means by which tosynthesize or obtain the desired compounds.

Another aspect of the invention comprises a compound used in the methodof the present invention as a pharmaceutical composition.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically active agent” means anysubstance or compound suitable for administration to a subject andfurnishes biological activity or other direct effect in the treatment,cure, mitigation, diagnosis, or prevention of disease, or affects thestructure or any function of the subject. Pharmaceutically active agentsinclude, but are not limited to, substances and compounds described inthe Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15,2009) and “Approved Drug Products with Therapeutic EquivalenceEvaluations” (U.S. Department Of Health And Human Services, 30^(th)edition, 2010), which are hereby incorporated by reference.Pharmaceutically active agents which have pendant carboxylic acid groupsmay be modified in accordance with the present invention using standardesterification reactions and methods readily available and known tothose having ordinary skill in the art of chemical synthesis. Where apharmaceutically active agent does not possess a carboxylic acid group,the ordinarily skilled artisan will be able to design and incorporate acarboxylic acid group into the pharmaceutically active agent whereesterification may subsequently be carried out so long as themodification does not interfere with the pharmaceutically active agent'sbiological activity or effect.

The compounds used in the method of the present invention may be in asalt form. As used herein, a “salt” is a salt of the instant compoundswhich has been modified by making acid or base salts of the compounds.In the case of compounds used to treat an infection or disease caused bya pathogen, the salt is pharmaceutically acceptable. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as phenols. The salts can bemade using an organic or inorganic acid. Such acid salts are chlorides,bromides, sulfates, nitrates, phosphates, sulfonates, formates,tartrates, maleates, malates, citrates, benzoates, salicylates,ascorbates, and the like. Phenolate salts are the alkaline earth metalsalts, sodium, potassium or lithium. The term “pharmaceuticallyacceptable salt” in this respect, refers to the relatively non-toxic,inorganic and organic acid or base addition salts of compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, or byseparately reacting a purified compound of the invention in its freebase or free acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The compounds of the present invention may also form salts with basicamino acids such a lysine, arginine, etc. and with basic sugars such asN-methylglucamine, 2-amino-2-deoxyglucose, etc. and any otherphysiologically non-toxic basic substance.

As used herein, “administering” an agent may be performed using any ofthe various methods or delivery systems well known to those skilled inthe art. The administering can be performed, for example, orally,parenterally, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery, subcutaneously, intraadiposally, intraarticularly,intrathecally, into a cerebral ventricle, intraventicularly,intratumorally, into cerebral parenchyma or intraparenchchymally.

The compounds used in the method of the present invention may beadministered in various forms, including those detailed herein. Thetreatment with the compound may be a component of a combination therapyor an adjunct therapy, i.e. the subject or patient in need of the drugis treated or given another drug for the disease in conjunction with oneor more of the instant compounds. This combination therapy can besequential therapy where the patient is treated first with one drug andthen the other or the two drugs are given simultaneously. These can beadministered independently by the same route or by two or more differentroutes of administration depending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the animal or human. The carrier maybe liquid or solid and is selected with the planned manner ofadministration in mind. Liposomes are also a pharmaceutically acceptablecarrier as are slow-release vehicles.

The dosage of the compounds administered in treatment will varydepending upon factors such as the pharmacodynamic characteristics of aspecific chemotherapeutic agent and its mode and route ofadministration; the age, sex, metabolic rate, absorptive efficiency,health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment being administered; thefrequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds used in the method of the presentinvention may comprise a single compound or mixtures thereof withadditional antitumor agents. The compounds can be administered in oraldosage forms as tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. The compounds may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, or introduced directly, e.g. byinjection, topical application, or other methods, into or topically ontoa site of disease or lesion, all using dosage forms well known to thoseof ordinary skill in the pharmaceutical arts.

The compounds used in the method of the present invention can beadministered in admixture with suitable pharmaceutical diluents,extenders, excipients, or in carriers such as the novel programmablesustained-release multi-compartmental nanospheres (collectively referredto herein as a pharmaceutically acceptable carrier) suitably selectedwith respect to the intended form of administration and as consistentwith conventional pharmaceutical practices. The unit will be in a formsuitable for oral, nasal, rectal, topical, intravenous or directinjection or parenteral administration. The compounds can beadministered alone or mixed with a pharmaceutically acceptable carrier.This carrier can be a solid or liquid, and the type of carrier isgenerally chosen based on the type of administration being used. Theactive agent can be co-administered in the form of a tablet or capsule,liposome, as an agglomerated powder or in a liquid form. Examples ofsuitable solid carriers include lactose, sucrose, gelatin and agar.Capsule or tablets can be easily formulated and can be made easy toswallow or chew; other solid forms include granules, and bulk powders.Tablets may contain suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Examples of suitable liquid dosage formsinclude solutions or suspensions in water, pharmaceutically acceptablefats and oils, alcohols or other organic solvents, including esters,emulsions, syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents. Oral dosage forms optionally contain flavorants andcoloring agents. Parenteral and intravenous forms may also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen.

Techniques and compositions for making dosage forms useful in thepresent invention are described in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents,coloring agents, flavoring agents, flow-inducing agents, and meltingagents. For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Suitable binders include starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds used in the method of the present invention may also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids suchas lecithin, sphingomyelin, proteolipids, protein-encapsulated vesiclesor from cholesterol, stearylamine, or phosphatidylcholines. Thecompounds may be administered as components of tissue-targetedemulsions.

The compounds used in the method of the present invention may also becoupled to soluble polymers as targetable drug carriers or as a prodrug.Such polymers include polyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds andpowdered carriers, such as lactose, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as immediate release products or as sustained releaseproducts to provide for continuous release of medication over a periodof hours. Compressed tablets can be sugar-coated or film-coated to maskany unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, asuitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water-soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobactene.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The compounds used in the method of the present invention may also beadministered in intranasal form via use of suitable intranasal vehicles,or via transdermal routes, using those forms of transdermal skin patcheswell known to those of ordinary skill in that art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill generally be continuous rather than intermittent throughout thedosage regimen.

Parenteral and intravenous forms may also include minerals and othermaterials such as solutol and/or ethanol to make them compatible withthe type of injection or delivery system chosen.

The compounds and compositions of the present invention can beadministered in oral dosage forms as tablets, capsules, pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions. Thecompounds may also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, or introduceddirectly, e.g. by topical administration, injection or other methods, tothe afflicted area, such as a wound, including ulcers of the skin, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts.

Specific examples of pharmaceutically acceptable carriers and excipientsthat may be used to formulate oral dosage forms of the present inventionare described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975.Techniques and compositions for making dosage forms useful in thepresent invention are described-in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, powders, and chewing gum; or in liquid dosageforms, such as elixirs, syrups, and suspensions, including, but notlimited to, mouthwash and toothpaste. It can also be administeredparentally, in sterile liquid dosage forms.

Solid dosage forms, such as capsules and tablets, may be enteric-coatedto prevent release of the active ingredient compounds before they reachthe small intestine. Materials that may be used as enteric coatingsinclude, but are not limited to, sugars, fatty acids, proteinaceoussubstances such as gelatin, waxes, shellac, cellulose acetate phthalate(CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate (hypromellose acetate succinate),polyvinyl acetate phthalate (PVAP), and methyl methacrylate-methacrylicacid copolymers.

The compounds and compositions of the invention can be coated ontostents for temporary or permanent implantation into the cardiovascularsystem of a subject.

Variations on those general synthetic methods will be readily apparentto those of ordinary skill in the art and are deemed to be within thescope of the present invention.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

The following materials and methods are used to test the compounds ofthe present invention.

Strains, Media and Reagents

A series of fungal clinical isolates and reference strains were used inthis study. This includes Cryptococcus neoformans, Cryptococcus gattii,Candida albicans, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida guiliiermondii, Aspergilius fumigatus, Rhizopusoryzae, Blastomyces dermatitis, Histoplasma capsulatum, Coccidioidesspp. Paecilomyces variotii, Pneumocystis murina, and, Pneumocystisjiroveci. Escherichia coli DH5-α and Pseudomonas aeruginosa were alsoused. Yeast Peptone Dextrose (YPD), Yeast Nitrogen Base (YNB), LuriaBertani (LB), Roswell Park Memorial Institute (RPMI) or DulbeccoModified Eagle Medium (DMEM) were purchased from Invitrogen LifeTechnologies and used as described. Fluconazole, Amphotericin B,Dexamethasone, Cyclophosphamide, Tunicamycin were purchased fromSigma-Aldrich, St Louis, Mo. Caspofungin and Posaconazole were obtainedfrom Merck, Rahway, N.J. Voriconazole was obtained from Pfizer, ReyBrook, N.Y.

In Vivo Labeling with Tritiated Palmitate (³H Palmitate)

Labeling fungal cells. C. neoformans cells were grown in YNB (pH 7.4) at37° C. in presence of 5% CO₂ for 16 hrs. Cells were centrifuged for 10min at 3,000 rpm at room temperature. Supernatant was removed and thecell pellet was suspended and counted. Next, 900 μL containing 5×10⁸ C.neoformans cells were placed into a 15 ml round bottom Corningcentrifuge tube. Then, 100 μL of different concentrations of compounddiluted in YNB containing 0.1% DMSO was added resulting in finalconcentrations of 0.25, 1 and 4 μg/ml, or 0.075, 0.3, 1.2 μg/ml,respectively. Tubes were incubated at in a shaker incubator at 225 rpmat 37° C. in the presence of 5% CO₂ for 4 hours. Then, 30 μCi/ml of ³Hpalmitate (PerkinElmer, Waltham, Mass.) was added to the culture andincubated for additional 2 hours. Cells without the drug were includedas negative control. The cells were then pelleted and washed once withdistilled sterile water and suspended in 1.5 ml of Mandala lipidextraction buffer. The lipids were extracted by the methods of Mandala,(Mandala, S. M. et al. 1997) and Bligh and Dyer followed by methanolicbased-hydrolysis as previously described (Bligh, E. G. & Dyer, W. J.1959). The tube was flushed with nitrogen gas and the samples dried in aSPD210 SpeedVac system (ThermoFisher Scientific, Waltham, Mass. Thedried lipids were resuspended in 30 μL of 1:1 methanol:chloroform andloaded on thin layered chromatography (TLC) silica gel 60 (EDMMillipore, Billerica, Mass.). Glucosylceramide (GlcCer) standard fromsoybean (Avanti Polar Lipids, Alabaster, Ala.) was added in a separatelane as control. The sample was resolved in a tank containing achloroform:methanol:water (65:25:4) as the mobile phase. The TLC plateswere then dried, exposed to iodine fume for the identification of theGlcCer standard band, which was marked. The TLC plate was then enhancedby spraying with ENHENCER (PerkinElmer) exposed to X-Ray film at −80° C.for 72 hours and the film was developed.

Labeling mammalian cells. The murine macrophage cell line J774.16 wasmaintained in Dulbecco Minimum Eagle Medium (DMEM) containing 10% FetalBovine Serum (FBS) and 1% Pen-strep by regular seeding. Cell at adensity of 5×10⁶ cells/ml of passage 8 were cultured in a 6 well cultureplate for 14 hours to achieve adherence. Compound at the sameconcentrations used for fungal cells (see above) were added to the platefor 4 hours. Then, 30 μCi/mL of ³H palmitic acid was added and the platewas further incubated for 2 hrs. Labeled J774.16 but untreated cellswere included as control. The cells were harvested by the addition of0.05% trypsin-EDTA and scraping with cell scrapper and washed once withPBS and dissolved in 2 ml methanol and 1 ml chloroform. Lipids wereextracted by the method of Bligh and Dyer followed by base hydrolysis.The samples were flushed with nitrogen and dried in SpeedVac. Driedlipids were suspended in 30 μL of 1:1 methanol:chloroform and loaded ona TLC plate with GlcCer as standard.

In Vitro Susceptibility Testing

Minimal inhibitory concentration (MIC) was determined following themethods of the Clinical Laboratory Standards Institutes (CLSI) withmodifications. MIC studies used either RPMI or YNB medium (pH 7.0, 0.2%glucose) buffered with HEPES. HEPES was used instead of MOPS becauseMOPS totally inhibits the activity of the compound. The compound wasserially diluted from 32 to 0.03 μg/ml or 19 to 0.02 μg/ml respectivelyin a 96 well plate with the respective medium. The yeast inoculum wasprepared as described in the CLSI protocol M27-A3 guidelines. Plateswere incubated at 37° C. and in the presence of 5% CO₂ for 24-96 hours.Against all fungal isolates used in the initial susceptibility screen,the MICs were determined as the lowest concentration of the drug thatinhibited 50% of growth compared to the control. MIC80 and MIC100, whosedrug concentrations inhibited 80% and 100% growth compared to thecontrol respectively, were also determined. For antibacterial activity,E. coli DH5a and P. aeruginosa PA14 were grown overnight in LuriaBertani (LB) broth at 30° C. The cells were washed with PBS and counted.Then, 300 μL from 2×10⁸ cells/mL was spreaded onto LB agar plate using ahockey stick glass spreader. The plate was dried, and wells were punchedout using a cut tip. Fifty microliters of different drug concentrationwas added to the well. The plate was then incubated at 30° C. for 24hours.

In Vitro Testing Against P. marina and P. jiroveci

Cryopreserved and characterized P. carinii isolated from rat lung tissue(Pc 08-4 #45) was distributed into triplicate wells of 48-well plateswith a final volume of 500 μL and a final concentration of 5×10⁷nuclei/ml. Control dilutions were added and incubated at 37° C. At 24,48, and 72 hours, 10% of the well volume was removed and the ATP contentwas measured using Perkin Elmer ATP-liteM luciferin-luciferase assay.The luminescence generated by the ATP content of the samples wasmeasured by a spectrophotometer (PolarStar Optima BMG, Ortenberg,Germany). A sample of each group was examined microscopically on thefinal assay day of the assay to rule out the presence of bacteriacontamination.

In Vitro Killing Assay

From an overnight culture, C. neoformans cells were washed in PBS,resuspended in YNB buffered with HEPES at pH 7.4. Cells were counted and2×10⁴ cells were incubated with either 1, 2 or 4 μg/ml of compound in afinal volume of 10 ml. Tubes were then incubated at 37° C. in thepresence of 5% CO₂ on a rotary shaker at 200 rpm. At the illustratedtime points, aliquots were taken and diluted and 100 μL was plated ontoyeast peptone dextrose (YPD) plates. YPD plates were incubated in a 30°C. incubator and, after 72 hours, colony forming units (CFU) werecounted and recorded.

Intracellular Effect

To assess whether the compound will be effective against intracellularC. neoformans, we first incubated J774.16 macrophages with C. neoformanscells at a 1:20 ratio in presence of opsonins (complement and antibodymAb 18B7 against the cryptococcal capsular antigen). After 2 hours ofincubation, about 60-80% of macrophages have at least one C. neoformanscell internalized. At this time, wells were washed to removeextracellular fungal cells and fresh DMEM medium without serum andwithout mAb 18B7 but containing different concentrations of compound wasadded. Plates were incubated at 37° C. and 5% CO₂. At selected timepoints, 0, 6, 12 and 24 hours, extracellular cells were collected bywashing and plated onto YPD for CFU counting of extracellular cells.Then, macrophages containing C. neoformans were lysed, collected andserial dilutions were plated onto YPD for CFU counting of intracellularfungal cells.

Synergistic Assay

Synergistic activity was assayed by calculating the fractionalinhibitory index (FIC) as previously described (Del Poeta, M. et al.2000). Briefly, in a 96 well plate, the compound was serially dilutedfrom 16 to 0.015 μg/ml (II dilutions) whereas drug B (e.g., eitherFluconazole, Amphotericin B, Caspofungin, or Tunicamycin) was seriallydiluted from 12 to 0.19 μg/ml, 5 to 0.078 μg/ml, 70 to 1.09 μg/ml, and 6to 0.09 μg/ml (7 dilutions), respectively. The FIC was defined as: [MICcombined/MIC Drug A alone]+[MIC combined/MIC Drug B alone].

Resistance Assay

To see whether incubation with the drugs will induce resistance, C.neoformans cells were passaged daily in sub-MIC drug concentrations.Briefly, from an overnight culture, C. neoformans cells were washed withPBS, resuspended in YNB buffered with HEPES at pH 7.4 and counted. Then,10⁶ cells were incubated with 0.5, 0.25 or 0.125 μg/ml of compound or0.15, 0.075 and 0.037 μg/ml of compound in 1 ml final volume. Tubeswithout the drug served as negative control. Tubes with Fluconazole(0.5, 1 and 2 μg/ml) served as positive control. The cells were grown at37° C. in the presence on 5% CO₂ on a rotary shaker at 200 rpm. Every 24hours, the cells were pelleted by centrifugation, washed with PBS, andresuspended in YNB, and 10^(t) cells were transferred into a fresh drugtube and incubated as above. These daily passages were continued for 15days. Cell aliquots were collected on day 0 (before any drug exposure),5, 10, 15, and MIC was determined using the microbroth dilution assay asdescribed above.

Animal Studies for Cryptococcosis

For survival studies, 4-week old CBA/J female mice (Jackson Laboratory,Bar Harbor, Me.) were used. Ten mice per treatment or control group wereused. Mice were infected by nasal inoculation of 20 μL containing 5×10⁵cells of C. neoformans H99 strain. Treated mice received anintraperitoneal injection of 1.2 mg/kg/day of compound in 100 μL finalvolume of PBS containing 0.4% DMSO. Untreated mice, received 100 μL ofPBS/0.4% DMSO. Mice were feed ad-libitum and monitored closely for signof discomfort and meningitis. Mice showing abnormal gait, lethargic,tremor, significant loss of body weight or inability to reach water orfood were sacrificed and survival counted from that day. At the end ofthe survival study, tissue burden culture was performed in mice thatsurvived the infection. Mice were sacrificed, and their organs wereextracted, and homogenized in 10 ml sterile PBS using a homogenizer(Stomacher80, Cole-Parmer, Vernon Hills, Ill.). Organ homogenates wereserially diluted 1:10 in PBS and 100 μL was plated on YPD agar platesand incubated at 30° C. for 72 hours for CFU count. For histopathology,extracted organs were fixed in 10% formalin before paraffin sectioningand staining with either Hematoxylin-Eosin or Mucicarmine. Images weretaken at 40× in a Zeiss Axio Observer in brightfield mode.

Animal Studies for Pneumocystosis

For survival studies, C3H/HeN mice ordered from the National CancerInstitute (Bethesda, Md.) were used. Mice were infected with P. murinapneumonia through exposure to mice with a fulminant P. murina infection(seed mice). These mice were immune suppressed by the addition ofdexamethasone at 4 mg/liter to the drinking water. Sulfuric acid at 1ml/liter was also added to the drinking water for disinfection. The seedmice are rotated within the cages for 2 weeks and then removed. Afterthe mice had developed a moderate infection level (approximately 5weeks), they were divided into a negative control group (controlsteroid), positive control group (trimethoprim/sulfamethoxazole) andtreatment groups (compound). Twelve mice were used in each group.Compound was administered intraperitoneally or by oral gavage on amg/kg/day basis for up to 3 weeks. The dose, route, and frequency ofadministration varied depending on the agent being tested. At the end ofthe treatment, mice were sacrificed and processed for analysis. Slideswere made from the lung homogenates at different dilutions and stainedwith Diff-Quik to quantify the trophic forms and Cresyl Echt violet toquantify the asci. Additional group of mice were selectively depleted oftheir CD4+ lymphocytes by antibody treatment with 300 μg of GK 1.5antibody (Biovest International, Minneapolis, Minn.) administeredintraperitoneally 3 times on days 1, 3, and 7. After this initialtreatment, the mice were infected by exposure to P. murina infectedmice. Mice then were treated with 100 μg of GK 1.5 antibodyintraperitoneally once a week for 6 weeks. Mice were then treated with1.25 or 12.5 mg/kg/day of 1 for 14 days while continuing the GK1.5treatment. Control mice received vehicle.

Animal Studies for Candidiasis

For survival studies, 8-week old CBA/J female mice (Jackson Laboratory)were used. Eight mice per treatment or control group were used. Micewere infected by intravenous inoculation of 100 μL containing 1×10⁵cells of Candida albicans SC-5314 strain. Treated mice received anintraperitoneal injection of 1.2 mg/kg/day of compound in 100 μL finalvolume of PBS containing 0.4% DMSO. Untreated mice, received 100 μL ofPBS/0.4% DMSO. Mice were feed ad-libitum and monitored closely for signof discomfort. At the end of the survival study, tissue burden culturewas performed in mice that survived the infection. Mice were sacrificedand their organs were extracted and homogenized in 10 ml sterile PBSusing homogenizer. Organ homogenates were diluted 10 times in PBS, and100 μL was plated on YPD agar plates and incubated at 30° C. for 72hours for CFU count.

Toxicity

In vitro. The murrine macrophage cell line J774.16 was maintained inDMEM containing 10% FBS and 1% Pen-strep. At passage #7, 10⁵ cells/wellin DMEM containing 10% FBS was transferred into 96 well plates andcultured for 14 hours for the cells to adhere to the wells. The compoundwas added to the cells at concentration ranging from 0.1 to 100 μg/ml.The wells without the drug served as control. The plate was incubated at37° C. in the presence of 5% CO₂. After 12 or 24 hours, the supernatantwas removed and 50 μL of 5 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)solution in PBS was added to each well and plates incubated for 4additional 4 hours. The formazan crystal formed inside the cell wasdissolved by adding 50 μL of isopropanol containing 0.1N HCL. Theoptical density was measured at 570 nm.

To determine whether the compound's toxicity was enhanced bycorticosteroids, a separate set of J774.16 cells were incubated with 10or 100 μg/ml of Dexamethasone alone or combined with either 1, 5 and 10μg/ml of compound. After 24 hours, the MTT assay was performed asdescribed above.

In Vivo.

Mice toxicity studies were performed using 4-week old CBA/J female micefrom Jackson Laboratory. Five mice received 1.2 mg/kg/day of compoundfor 60 days. Three control mice received a solvent injection per day. At60-day, blood was collect in two tubes: one with K₂EDTA and the otherwithout K₂EDTA to allow blood clotting. The blood clot was thencentrifuged at 1500 rpm for 10 min, serum was collected and analyzed forliver and kidney blood tests. The non-coagulated blood was used forhematocrit and blood cells analysis. These tests were done using MASCOT™HEMAVET 950FS (Drew Scientific Group, Dusseldorf, Germany).

Lipid Mass Spectrometry

For lipid analysis by mass spectrometry, fungal cells (C. neoformans orC. albicans) were grown in YNB and incubated with compound as explainedfor the in vivo labeling (except that tritiated palmitate was notadded), for 6 hrs. Samples without drug were included as control. Beforelipid extraction, lipid internal standards (C17 ceramide and C17sphingosine) were added. Lipids were then extracted following themethods of Mandala and Bligh and Dyer and one fourth of the sample wasaliquoted for determination of the inorganic phosphate. The remainder ofthe sample was subjected to base hydrolysis and then analyzed usingLC/MS. Results were normalized with the inorganic phosphate levels.

In Vitro Activity of Gcs1

For the in vitro Gcs1 assay, C. neoformans wild-type (WT) or the Δgcs1cells were grown in YPD broth overnight at 30° C. in a shaker incubator.Cells were washed with sterile water and then lysed by bead beating inpresence of glass bead and protease cocktail inhibitor, as described(Liberto, C. et al. 2001). Next, 800 μg of cell lysate was incubatedwith 0.3 mM C16 ceramide (C16-R—OH) and in the presence or absence ofcompound. The mixture was subjected to 3 cycles of sonication (20 sec)and vortexing (5 sec). Next, 8 μM of radiolabelled UDP-¹⁴C-Glucose(American Radiolabeled Chemical) was added and, after brief vortexing,the tubes were incubated at 37° C. for 45 min. The reaction was stoppedby adding 0.9 ml of 0.45% NaCl solution containing chloroform:methanol2:1. The organic phase was collected in a glass tube and flushed withnitrogen. The sample was dried and resuspended in chloroform:methanol1:1. Sample was then loaded on a TLC plate using bychloroform:methanol:water as the mobile phase.

Yeast Library Screening

Variomics Library:

The screening of the Saccharomyces cerevisiae genome-wide variomicslibraries for potential compound resistant clones was performed asdescribed previously (Huang, Z. et al. 2013) but with slightmodifications. About 6×10⁴ haploid cells was plated on solid SC-Uramedium buffered with HEPES at pH 7.0, which contained compound at aconcentration of 20 μM (˜7 μg/ml) and incubated at 30° C. for 3 days.

HIP-HOP Library:

The yeast deletion collection used here comprises of approximately 5900individually barcoded heterozygous diploid strains (HaploInsufficiencyProlifing) and ˜4800 homozygous diploid strains (HOmozygous deletionProfiling) (Pierce, S. E. et al. 2007). Pools of approximately equalstrain abundance were generated by robotically pinning (S and PRobotics, Ontario, Canada) each strain (from frozen stocks) onto YPDagar plates as arrays of 384 strains/plate. After two days of growth at30° C., colonies were collected from plates by flooding with YPD andaliquoted at optical density of 2 (at 600 nm). The fitness of eachstrain in each experimental pool was assessed as described (Pierce, S.E. et al. 2007). The dose of compound that resulted in 15% growthinhibition in BY4733 (the parent strain of the yeast deletioncollection) was determined by performing a dose response over the courseof 16 h of growth at 30° C. Screens of the homozygous deletioncollection were performed for 5 generations of growth in compound, andscreens of the Heterozygous deletion collection were collected following20 generations of growth. Cells were processed as described (Proctor, M.et al. 2011). Briefly genomic DNA was extracted from each sample,subjected to PCR to amplify the unique barcode identifiers and theabundance of each barcode was determined by quantifying the microarraysignal as described. A ranked list of all genes in the genome wasgenerated for each experiment and then compared using gene setenrichment analysis or GSEA according to Lee (Lee, A Y et al. 2014).

C6-NBD-Ceramide Staining

The Golgi apparatus of C. neoformans and C. albicans was stained withC6-NBD-ceramide using a previously described protocol (Kmetzsch, L. etal. 2011), based on the property that this fluorescent lipid accumulatesat the Golgi of either living or fixed cells (Pagano R. E. et al. 1989).Control or compound-treated (4 μg/ml) yeast cells were fixed with 4%paraformaldehyde in PBS. Cell suspensions were then washed with the samebuffer and incubated with C6-NBD-ceramide (20 mM) for 16 h at 4° C. Thecells were then incubated with bovine serum albumin (BSA, 1%) at 4° C.for 1 h to remove the excess of C6-NBD-ceramide. After washing with PBS,the cells were incubated with 10 μg/ml DAPI (Sigma-Aldrich, St. Louis,USA) for 30 min at room temperature. The cells were washed again withPBS and stained cell suspensions were mounted over glass slides asdescribed above and analyzed under an Axioplan 2 (Zeiss, Germany).

Statistical Analysis

Statistical analysis for survival studies was performed usingStudent-Newman-Keuls t test for multiple comparisons using INSTAT.Statistical analysis for tissue burden and for trophic form and ascicounts was performed using the analysis of variance (ANOVA). Additionalstatistic was performed using Student t test.

Comparison Studies

For survival studies, 4-week old CBA/J female mice (Jackson Laboratory,Bar Harbor, Me.) were used. Total of forty mice were infected by tailvein injection of 200 μL containing 10¹ cells of C. neoformans H99 andwere randomly separated into 5 groups (8 mice per group). Treatmentstarted within 2 hours of infection. The treated mice received anintraperitoneal injection of 1.2 mg/kg/day of compound and amphotericinB or 10 mg/kg/day of fluconazole in 100 μL final volume of PBScontaining 0.4% DMSO. Untreated mice, received 100 μL of PBS/0.4% DMSO.Mice were fed ad-libitum and monitored closely for sign of discomfortand meningitis. Mice showing abnormal gait, lethargy, tremor,significant loss of body weight, or inability to reach water or foodwere sacrificed and survival was counted until that day.

Sample Preparation for Transmission Electron Microscopy (TEM)

Sample preparation for Transmission electron Microscopy (TEM) wasperformed similar to the methods of Heung (Heung et al. 2005) with minormodifications. Briefly, C. neoformans (H99) were grown in YNB (pH=7.4)at 37° C. and 5% CO₂ and treated for 6 hours with compound (4 μg/mL),non-treated cells were also included as control. The cells were pelletedat 3000 rpm (1700 g) and fixed with 2% EM glutaraldehyde in PBS solutionfor 1 hour. Samples were then washed in PBS, placed in 1% osmiumtetroxide in 0.1M PBS, dehydrated in a graded series of ethyl alcoholand embedded in Embed812 resin. Ultrathin sections of 80 nm were cutwith a Leica EM UC7 ultramicrotome (Leica Microsystems Inc., BuffaloGrove, Ill.) and placed on uncoated mesh copper grids. Sections werethen counterstained with uranyl acetate and lead citrate and viewed witha FEI Tecnai12 BioTwinG2 electron microscope (FEI, Hillsboro, Oreg.)Transmission Electron Microscope (TEM). Digital images were acquiredwith an AMT XR-60 CCD Digital Camera system.

Pre-Screening

For the compound revertant screen, the drug-sensitive RYO0622 haploidstrain was used (Suzuki, Y., et al. 2011). To determine the IC₁₀₀ doseof compound (at which yeast cell growth is inhibited at 100% upon drugexposure), 20 ul of RYO0622 cells (at OD₆₀₀ 1⁻⁴) were plated on solidsynthetic complete (SC) media alone, with DMSO, or with a range ofcompound doses (0.2, 0.4, 0.8, 1.6 and 3.2 mM) in a 46-well plate. Theplate was incubated for 2 days at 30° C. in the dark.

Revertant Screening Assay

RYO0622 cells were cultured to mid-log phase (˜OD₆₀₀ 0.5) in liquid SCmedia before adjusting the cell density to 1×10⁶ cells/ml (equivalent toOD₆₀₀˜0.1). One ml of cells was plated on solid SC media containing DMSOsolvent control (0.26% v/v) or compound (at 0.4 mM IC100 dose) andincubated at 30° C. in the dark. A lawn of cells grew on the solventcontrol, while only a single compound-resistant colony was identifiedafter 9 days. Longer incubation did not result in the appearance offurther resistant clones. To confirm compound resistance, singlecolonies isolated from the compound containing SC media were plated ontofresh solid SC medium containing 0.4 mM compound and incubated for 2days at 30° C. in the dark. Robust compound-resistant cells were seen.

Yeast Genomic DNA Preparation

Genomic DNA was extracted from RYO0622 and compound-resistant cellsusing the Puregene kit (Qiagen), according to the manufacturer'sinstructions.

Next-Generation Sequencing of Compound-Resistant RYO0622

Genomic DNA was quantified using Qubit fluorometry (Life Technologies)and diluted for sequencing library preparation using Nextera XT librarypreparation kit according to the manufacturer's instructions (Illumina).Libraries were pooled and sequenced on a single MiSeq lane, generatingpaired-end 150 bp reads.

Mapping & Variant Calling

Raw FASTQ paired-end reads for the parent (RYO0622) and the revertantwere independently aligned to NCBI sacCer3(genbank/genomes/Eukaryotes/fungi/Saccharomyces_cerevisiae/SacCer_Apr2011) reference genome using bwa memv0.7.4-r385 with the −M flag to mark shorter split hits as secondary forcompatibility with Picard (Li, H. & Durbin, R. 2009). Resultant SAMfiles were converted to BAM format using samtools v1.1 and sorted bycoordinate using Picard v1.96 (SortSam) (http://picard.sourceforge.net).PCR duplicate reads were filtered out using Picard MarkDuplicates(10.24% estimated duplication) and indexed using Picard BuildBamIndex.To call single nucleotide variants (SNVs), we ran the GATK UnifiedGenotyper v2.1-8 (McKenna, A., et al. 2010) with the NCBI sacCer3reference genome, stand_call_conf=30, and stand_emit_conf=10 (DePristo,M. A., et al. 2011). The ploidy parameter was set to 1 since the parentand revertant are in haploid state. Since a database of known indels andknown SNPs was not available, we did not perform re-alignment aroundknown indels and quality score recalibration.

TEM.

Sample preparation for Transmission electron Microscopy (TEM) wasperformed similar to the methods of Hueng et al. with minormodifications (Heung, L. J. et al 2005). Briefly, C. neoformans (H99)were grown in YNB (pH=7.4) at 37° C. and 5% CO₂ and treated for 6 hourswith compound (4 μg/mL), non-treated cells were also included ascontrol. The cells were pelleted at 3000 rpm (1700 g) and fixed with 2%EM glutaraldehyde in PBS solution for 1 hour. Samples were then washedin PBS, placed in 1% osmium tetroxide in 0.1M PBS, dehydrated in agraded series of ethyl alcohol and embedded in Embed812 resin. Ultrathinsections of 80 nm were cut with a Leica EM UC7 ultramicrotome (LeicaMicrosystems Inc., Buffalo Grove, Ill.) and placed on uncoated meshcopper grids. Sections were then counterstained with uranyl acetate andlead citrate and viewed with a FEI Tecnai12 BioTwinG2 electronmicroscope (FEI, Hillsboro, Oreg.) Transmission Electron Microscope(TEM). Digital images were acquired with an AMT XR-60 CCD Digital Camerasystem.

Generation of Compound-Resistant Strains.

For the generation of compound-resistant strains, the drug-sensitive S.cerevisiae RYO0622 haploid strain was used (Suzuki, Y. et al. 2011).Prescreening studies were performed to determine the IC₁₀₀ dose ofcompound for this strain (the 100% inhibitory concentration [IC₁₀₀] atwhich 100% yeast cell growth is inhibited upon drug exposure). For thisscreening, 20 μl of RYO0622 cells (at an OD₆₀₀ of 10⁻⁴) were plated onsolid synthetic complete (SC) medium alone or with DMSO or with variouscompound concentrations (67, 133, 266, 533, and 1,066 μg/ml) in a48-well plate. The plates were incubated for 2 days at 30° C. in thedark. These studies revealed an IC₁₀₀ dose of 133 μg/ml. Screening forthe compound-resistant mutants was performed by growing the RYO0622cells to mid-log phase (OD₆₀₀ of 0.5) in liquid SC medium beforeadjusting the cell density to 1×10⁶ cells/ml (equivalent to an OD₆₀₀ of0.1). One milliliter of cells was plated on solid SC medium containingDMSO solvent control (0.26% [vol/vol]) or compound (133 μg/ml IC₁₀₀dose) and incubated at 30° C. in the dark. A lawn of cells grew on thesolvent control, while seven compound-resistant colonies were identifiedafter 9 days. Longer incubation did not result in the appearance offurther resistant colonies. To confirm compound resistance, singlecolonies isolated from the compound-containing SC medium were platedonto fresh solid SC medium containing 133 μg/ml compound and incubatedfor 2 days at 30° C. in the dark. Robust compound-resistant cells wereseen.

Next-Generation Sequencing of Compound-Resistant Strains.

Genomic DNA was extracted from RYO0622 and compound-resistant cellsusing a standard yeast DNA extraction protocol (Hoffman, C. S. et al.1987). Genomic DNA samples were quantified using Qubit fluorometry (LifeTechnologies) and diluted for sequencing library preparation using aNextera XT library preparation kit according to the manufacturer'sinstructions (Illumina, San Diego, Calif.). For the initial round ofsequencing, individual sequencing libraries were prepared for the parentand a single compound-resistant clone. These libraries were pooled andsequenced on a single MiSeq lane (Illumina), generating paired-end150-bp reads. Further compound-resistant colonies were obtained in asecond screen, and their DNAs were pooled at equal concentrations beforepreparation of a single sequencing library for the pool. This pool wassequenced alongside a new library for the parent strain on a singleHiSeq 2500 lane (Illumina), generating paired-end 100-bp reads.

Mapping and Variant Calling.

Raw FASTQ paired-end reads for the parent (RYO0622) and thecompound-resistant pool were independently aligned to the NCBI sacCer3reference genome using bwa mem v0.7.4-r385 (Li, R., Yu, C. et al. 2009)with the −M flag to mark shorter split hits as secondary forcompatibility with Picard. Resultant SAM files were converted to BAMformat using samtools v1.1 and sorted by coordinate using Picard v1.96(SortSam). PCR duplicate reads were filtered out using PicardMarkDuplicates and indexed using Picard BuildBamIndex. To call singlenucleotide variants (SNVs), the GATK Unified Genotyper v2.1-8 (McKenna,A. et al. 2010) was ran with the NCBI sacCer3 reference genome,stand_call_conf=30, and stand_emit_conf=10 (DePristo, M. A. et al.2011). The ploidy parameter was set at 1, since the parent and resistantstrains are in haploid state. Realignment around known indels andquality score recalibration was not performed, since a database of knownindels and known single nucleotide polymorphisms (SNPs) is notavailable.

Validation of Compound-Resistant Yeast Mutants.

Four yeast genes (ALP5, COS111, MKK1, and STE2) were selected based onthe high-quality variant calls present in the compound-resistant pool.To confirm compound resistance, the individual haploid Δap15, Δcos111,Δmkk1 and Δste2 deletion mutants were assayed for growth fitness aftertreatment with compound. Unrelated drug controls, including methylmethane sulfonate (MMS) (cytotoxic) and fluconazole (antifungal) wereassayed in parallel. Strains were cultured to mid-log phase (OD₆₀₀ of˜0.5) in liquid YPD medium before adjusting the cell density to an OD₆₀₀of 0.0625 with YPD medium. The cells were transferred to 96-well platescontaining 100 μl of YPD with DMSO solvent control (2% [vol/vol]),compound (6 to 733 μg/ml), MMS (10 μg/ml to 625 μg/ml), or fluconazole(2 to 306 μg/ml) and incubated at 30° C. for 24 h. The fitness ofindividual strains was measured using a spectrophotometer plate reader(Tecan GENios, Chapel Hill, N.C.) to read OD₆₀₀ over 24 h as a proxy forcell growth. Relative growth inhibition was calculated by the averagerate after normalizing the OD₆₀₀ values in drug wells against the DMSOcontrol wells on each assay plate.

Example 1. Synthesis

General Synthesis of Compounds

The benzaldehyde (1 mmol, 2 ml ethanol) and the benzohydrazide (1 mmolin 2 ml hot ethanol) were combined. The product generally crystallizewithin seconds. After 30 minutes at room temperature the product wascollected by filtration (Yield: 80 to 95%). Homogeneity of the productwas confirmed by thin layer chromatography (TLC) on silicagel F₂₅₄(Merck KGaA, Darmstadt, Germany) in two different solvent systemsbenzene/acetic acid 9:1 v/v and hexane/ethylacetate 1:3 v/v. Ifimpurities were present the product was recrystallized from ethanol.Alternatively, the reaction proceeds for 24 h at 4° C. and the solventwas completely evaporated and the product crystallized from ethylacetate. Products were analyzed by TLC as described above.

Synthesis of 1

To a solution of 2-methylbenzoic hydrazide (0.66 mmol),2-hydroxy-5-bromobenzaldehyde (0.72 mmol) in methanol (3 mL) was added 3drops of glacial acetic acid. The reaction mixture was stirred at roomtemperature overnight. Addition of water to the reaction mixtureresulted in precipitation of the product, which was filtered, washedwith water and dried to give pure product as white solid. (95% yield);¹H NMR (500 MHz, DMSO-d6) δ 2.38 (s, 3H), 6.90 (d, 1H, J=8.8 Hz),7.28-7.32 (m, 3H), 7.37-7.43 (m, 2H), 7.47 (d, 1H, J=7.5 Hz), 7.78 (s,1H), 8.47 (s, 1H), 11.19 (s, 1H), 12.05 (s, 1H). Compound 2, 13 and 17were synthesized by an analogous method using the appropriate startingmaterials for each.

Example 2. PK/PD: Killing Curve Versus MIC K_(app) Coefficient Analysis

Compound MIC K_(app)* A549 SI_(MIC) 1 1 0.024 32 32 2 0.25 −0.142 64 6413 0.06 −0.428 >128 4266.6 17 0.5 0.077 16 32 *K_(app) = K₀ − K_(i) (C −C_(i))

Example 3. Kill Characteristics of 1 and 2

Compound 1 killed C. neoformans in a concentration dependent manner (seeFIG. 1A). Compound 2 also killed C. neoformans in a time dependentmanner (see FIG. 1B).

Example 4. Fungicidal Data

TABLE 1 Killing assay Structure IC₈₀(μg/mL) 48H (μg/mL) A549 HepG2SI-A549/MIC SI-HepG2/MIC

0.25 Fungistatic 32 32 128 128

0.25 0.5 16 16 64 64

1 1 32 16 32 16

1 0.5 32 32 32 32

0.25 0.25 4 8 16 32

0.25 1 64 64 256 256

1 0.5 72 32 72 32 *Units are μg/ml for A549, HepG2, SI-A549/MIC andSI-HepG2/MIC.

TABLE 2 Killing assay Structure IC₈₀(μg/mL) 48H (μg/mL) A549 HepG2SI-A549/MIC SI-HepG2/MIC

0.25 0.12 59 32 236

1 0.5 >64 64 128 64

0.5 0.25 32 32 64 64

0.12 0.5 16 >32 133.3 533.3

0.25 0.25 32 16 128 64

0.5 0.25 >32 9 128 18

0.06 0.12 >32 >64 1066 2133.3 *Units are μg/ml for A549, HepG2,SI-A549/MIC and SI-HepG2/MIC.

TABLE 3 Killing assay Structure IC₈₀(μg/mL) 48H (μg/mL) A549 HepG2SI-A549/MIC SI-HepG2/MIC

0.5 0.25 16 16 32 32

0.06 Fungistatic 16 32 266.6 533.3

0.12 0.25 32 16 266.6 133.3

0.06 Fungistatic 32 32 533.3 533.3

0.12 0.12 >32 59 533.3 491.66

0.25 1 16 32 64 128 *Units are μg/ml for A549, HepG2, SI-A549/MIC andSI-HepG2/MIC.

Example 5. Administration of the Compound

An amount of the compound of the present invention is administered to asubject afflicted with a fungal infection. The amount of the compound iseffective to treat the subject.

An amount of the compound of the present invention is administered to asubject afflicted with a fungal infection. The amount of the compound iseffective to treat the subject by inhibiting sphingolipid synthesis inthe fungus without substantially inhibiting sphingolipid synthesis inthe subject.

An amount of the compound of the present invention in combination withan anti-fungal agent are administered to a subject afflicted with afungal infection. The amount of the compound and the agent are effectiveto treat the subject.

Example 6: Assessment of Efficacy of Compound as Add-on Therapy toAnti-Fungal Agents

The add-on therapy provides a synergistic effect, and allows for lowerdoses with reduced side effects and resistance.

Periodic administration of the compound of the present invention as anadd-on therapy for a subject afflicted with a fungal infection who isalready receiving treatment with an anti-fungal agent provides aclinically meaningful advantage and is more effective (provides at leastan additive effect or more than an additive effect) in treating thesubject than when the anti-fungal agent is administered alone (at thesame dose).

Periodic administration an anti-fungal agent as an add-on therapy for ahuman patient afflicted with a fungal infection who is already receivinga compound of the present invention provides a clinically meaningfuladvantage and is more effective (provides at least an additive effect ormore than an additive effect) in treating the subject than when thecompound is administered alone (at the same dose).

The add-on therapies also provide efficacy (provides at least anadditive effect or more than an additive effect) in treating the subjectwithout undue adverse side effects or affecting the safety of thetreatment. As compared to when each agent is administered alone:

1. The add-on therapy is more effective (provides an additive effect ormore than an additive effect) in killing the fungus; and/or

2. The add-on therapy is more effective (provides an additive effect ormore than an additive effect) in slowing the growth of the fungus.

Example 7: Synthesis and Characterization

Chemical Synthesis and Characterization of Aromatic Acylhydrazones ofthis Invention

4-Bromo-N′-(2-hydroxy-5-trifluoromethoxybenzylidene)benzohydrazide (A1)

To a solution of 4-bromobenzoic hydrazide (100 mg, 0.47 mmol),5-trifluoromethoxysalicylaldehyde (102 mg, 0.49 mmol) in methanol (2 mL)was added 3 drops of glacial acetic acid. The reaction mixture wasstirred at room temperature overnight. Addition of water to the reactionmixture resulted in precipitation of the product, which was filtered,washed with water and dried, to give pure product as white solid (93%yield); m.p. 213-214° C.; 1H NMR (500 MHz DMSO-de) δ 7.02 (d, 1H, J=9Hz), 7.30 (dd, 1H, J=8.9, 2.6 Hz), 7.64 (s, 1H), 7.77 (d, 2H, J=8.5 Hz),7.89 (d, 2H, J=8.5 Hz), 8.67 (s, 1H), 11.25 (s, 1H), 12.23 (s, 1H); ¹³CNMR (125 MHz DMSO-d₆) δ 117.73, 119.19, 120.21, 120.37, 121.22, 123.25,124.34, 125.83, 129.77, 131.60, 131.87, 140.67, 145.61, 156.06, 162.06;¹⁹F NMR (376 MHz DMSO-d₆) δ −57.30 (s, 3 F); HRMS (ESI) m/z calcd forC₁₅H₁₀BrF₃N₂O₃H⁺: 402.99, Found: 402.991 (Δ=−2.47 ppm).

The same procedure was followed for the rest of the compounds.

4-Bromo-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide (A2)

White solid (88% yield); m.p.>220° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.92-6.94 (m, 1H), 7.15 (td, 1H, J=8.6, 3.2 Hz), 7.44 (dd, 1H, J=9.4 3.1Hz), 7.76 (d, 2H, J=8.5 Hz), 7.89 (d, 2H, J=8.5 Hz), 8.63 (s, 1H), 10.94(s, 1H), 12.20 (s, 1H); ¹³C NMR (125 MHz DMSO-d₆) δ 113.61, 113.80,117.58, 117.64, 118.01, 119.74, 119.80, 125.80, 129.74, 131.58, 131.86,146.34, 153.57, 154.39, 156.26, 161.97; ¹⁹F NMR (376 MHz DMSO-d₆) δ−125.06 (s, 1 F); HRMS (ESI) m/z calcd for C₁₄H₁₀BrFN₂O₂H⁺: 336.9982,Found: 336.9996 (Δ=−3.95 ppm).

2,4-Dibromo-N′-(2-hydroxy-5-trifluoromethoxybenzylidene)benzohydrazide(A3)

White solid (99% yield); m.p. 170-172° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.91 (d, 1H, 40%, J=8.8 Hz), 7.01 (d, 1H, 60%, J=8.9 Hz), 7.16-7.17 (m,1H, 50%), 7.19-7.20 (1H, m, 20%), 7.30 (dd, 1H, 60%, J=9, 2.8 Hz), 7.40(d, 1H, 40%, J=8.2 Hz), 7.54 (d, 1H, 60%, J=8.2 Hz), 7.64 (s, 1H, 60%),7.69 (dd, 1H, 40%, J=8.2, 1.8 Hz), 7.74 (dd, 1H, 60%, J=8.2, 1.8 Hz),7.97 (s, 1H, 35%), 8.02 (s, 1H, 40%), 8.50 (s, 1H, 60%), 10.28 (s, 1H,40%), 11.03 (s, 1H, 60%), 12.18 (s, 1H, 63%), 12.20 (s, 1H, 34%); ¹³CNMR (125 MHz DMSO-d₆) δ 117.54, 117.73, 118.54, 119.06, 119.17, 119.86,119.94, 120.19, 120.61, 120.63, 121.09, 121.21, 123.78, 123.86, 124.56,130.15, 130.57, 130.80, 130.87, 134.01, 134.84, 136.08, 137.20, 140.69,140.70, 140.85, 145.36, 155.16, 155.96, 162.52, 168.45; ¹⁹F NMR (376 MHzDMSO-d₆) δ −57.31, −57.40; HRMS (ESI) m/z calcd for C₁₅H₉Br₂F₃N₂O₃H⁺:480.9005, Found: 480.9013 (Δ=−1.74 ppm).

2,4-Dibromo-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide (A4)

White solid (96% yield); m.p. 182-183° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.83 (dd, 1H, 40%, J=9, 4.7 Hz), 6.94 (dd, 1H, 60%, J=9, 4.7 Hz), 6.98(dd, 1H, 40%, J=9.4, 3.2 Hz), 7.06 (td, 1H, 40%, J=8.5, 3.3 Hz), 7.16(td, 1H, 60%, J=8.5, 3.3 Hz), 7.41 (d, 1H, 40%, J=8.2 Hz), 7.44 (dd, 1H,60%, J=9.4, 3.2), 7.54 (d, 1H, 60%, J=8.2 Hz), 7.70 (dd, 1H, 40%, J=8.2,1.9 Hz), 7.74 (dd, 1H, 60%, J=8.2, 1.9 Hz), 8.00 (s, 1H, 35%), 8.02 (s,1H, 55%), 8.27 (s, 1H, 40%), 8.47 (s, 1H, 60%), 9.90 (s, 1H, 40%), 10.71(s, 1H, 60%), 12.15 (s, 1H, 66%), 12.17 (s, 1H, 33%); ¹³C NMR (125 MHzDMSO-d₆) δ 111.98, 112.18, 113.20, 113.39, 117.44, 117.50, 117.58,117.65, 117.78, 117.97, 118.25, 118.43, 119.75, 119.81, 120.18, 120.24,120.63, 122.66, 123.76, 130.16, 130.66, 130.78, 130.86, 134.13, 134.82,136.08, 137.16, 141.81, 146.09, 152.80, 153.48, 154.32, 154.41, 156.18,156.27, 162.45, 168.29; ¹⁹F NMR (376 MHz DMSO-d₆) δ −124.76 (s, 1 F),−124.91 (s, 1 F); HRMS (ESI) m/z calcd for C₁₄H₉Br₂FN₂O₂H⁺: 414.9088,Found: 414.9095 (Δ=−1.7 ppm).

3-Difluoromethoxy-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (A5)

Yellow solid (90% yield); m.p. 166-168° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.91 (d, 1H, 90%, J=8.8 Hz), 6.95 (d, 1H, 10%, J=8.8 Hz), 7.18 (s, 1H,25%), 7.33 (s, 1H, 50%), 7.42-7.44 (m, 2H, 90%), 7.48 (s, 1H, 25%),7.54-7.56 (m, 2H, 10%), 7.61 (t, 1H, 100%, J=8 Hz), 7.72 (s, 1H, 90%),7.81-7.83 (m, 2H, 100%, 90%), 7.90 (s, 1H, 10%), 8.63 (s, 1H, 90%), 8.93(s, 1H, 10%), 11.13 (s, 1H, 10%), 11.20 (s, 1H, 90%), 12.22 (s, 1H,90%); ¹³C NMR (125 MHz DMSO-d₆) δ 110.48, 110.56, 114.26, 117.94,118.36, 118.68, 118.91, 120.57, 121.33, 122.26, 124.45, 130.23, 130.41,131.57, 133.70, 134.65, 135.49, 145.84, 150.94, 150.96, 156.41, 157.66,160.76, 161.83; ¹⁹F NMR (376 MHz DMSO-d₆) δ −82.16 (s, 2 F); HRMS (ESI)m/z calcd for C₁₅H₁₁BrF₂N₂O₃H⁺: 384.9994, Found: 384.9996 (Δ=−0.49 ppm).

3-Difluoromethoxy-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide

Yellow solid (99% yield); m.p. 143-147° C.; ¹H NMR (500 MHz DMSO-d₆) δ7.19 (s, 1H, 25%), 7.34 (s, 1H, 50%), 7.46 (d, 1H, 90%, J=8 Hz), 7.49(s, 1H, 25%), 7.63 (t, 1H, 100%, J=8 Hz), 7.73 (s, 1H, 100%), 7.83-7.96(m, 3H, 100%), 8.54 (s, 1H, 90%), 9.06 (s, 1H, 10%), 10.08 (s, 1H, 5%),11.99 (s, 1H, 10%), 12.60 (s, 1H, 90%), 12.64 (s, 1H, 95%); ¹³C NMR (125MHz DMSO-d₆) δ 110.44, 110.80, 111.25, 111.64, 114.22, 116.28, 118.05,118.34, 120.35, 120.92, 122.55, 124.53, 130.50, 132.14, 133.47, 134.02,135.69, 137.77, 147.49, 150.98, 151.00, 153.65, 154.71, 161.94, 163.98;¹⁹F NMR (376 MHz DMSO-d₆) δ −82.22 (s, 2 F); HRMS (ESI) m/z calcd forC₁₅H₁₀Br₂F₂N₂O₃H⁺: 462.9099, Found: 462.91 (Δ=−0.2 ppm).

4-Trifluoromethoxy-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (A7)

Beige solid (88% yield); m.p. 199-201° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.91 (d, 1H, J=8.8 Hz), 7.42-7.44 (m, 1H), 7.54 (d, 2H, J=8.3 Hz), 7.81(s, 1H), 8.07 (d, 2H, J=8.8 Hz), 8.61 (s, 1H), 11.21 (s, 1H), 12.25 (s,1H); ¹³C NMR (125 MHz DMSO-d₆) δ 110.47, 116.88, 118.68, 118.92, 120.83,120.97, 121.31, 123.02, 130.10, 130.28, 131.88, 133.69, 145.81, 150.74,156.42, 161.77; ¹⁹F NMR (376 MHz DMSO-d₆) δ −56.64; HRMS (ESI) m/z calcdfor C₁₅H₁₀BrF₃N—O₃H⁺: 402.99, Found: 402.9902 (Δ=−0.63 ppm).

2-Fluoro-4-trifluoromethyl-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide(A8)

Yellow solid (61% yield); m.p. 179-182° C.; ¹H NMR (400 MHz DMSO-d₆) δ6.80 (d, 1H, 30%, J=8.6 Hz), 6.90 (d, 1H, J=8.7 Hz), 7.29-7.31 (m, 2H,25%), 7.33-7.34 (m, 1H, 17%), 7.44 (dd, 1H, 80%, J=8.8, 2.6 Hz),7.72-7.76 (m, 2H, 75%), 7.81-7.93 (m, 2H, 100%), 8.29 (s, 1H, 33%), 8.51(s, 1H, 77%), 10.26 (s, 1H, 30%), 11.00 (s, 1H, 70%), 12.25 (s, 1H,30%), 12.27 (s, 1H, 70%); ¹³C NMR (100 MHz DMSO-d₆) δ 110.55, 113.87,114.12, 118.46, 118.68, 121.24, 121.64, 121.87, 126.70, 128.19, 129.99,131.54, 133.52, 133.96, 140.99, 145.98, 155.63, 156.38, 157.70, 159.21,160.21, 165.61; ¹⁹F NMR (376 MHz DMSO-d₆) δ −61.33 (s, 3 F), −61.46 (s,3 F), −111.26 (s, 1 F), −111.49 (s, 1 F); HRMS (ESI) m/z calcd forC₁₅H₉BrF₄N₂O₂H⁺: 404.9856, Found: 404.9864 (Δ=−1.84 ppm).

2-Fluoro-4-trifluoromethyl-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide(A9)

Yellow solid (74% yield); m.p. 192-195° C.; ¹H NMR (400 MHz DMSO-d₆) δ7.52 (s, 1H, 15%), 7.74 7.97 (m, 1H, 85%, 4H, 100%), 8.27 (s, 1H, 20%),8.46 (s, 1H, 80%), 10.2 (s, 1H, 25%), 12.34 (s, 1H, 75%), 12.53 (s, 1H,15%), 12.67 (s, 1H, 80%); ¹³C NMR (100 MHz DMSO-d₆) δ 110.57, 111.17,111.92, 113.96, 114.21, 120.89, 121.58, 121.69, 121.73, 122.27, 126.01,126.17, 130.49, 130.65, 131.66, 131.69, 132.84, 132.92, 133.16, 133.25,135.68, 135.96, 143.57, 148.06, 152.33, 153.62, 157.78, 159.35, 160.29,165.41; ¹⁹F NMR (376 MHz DMSO-d₆) δ −61.34 (s, 3 F), −61.50 (s, 3 F),−111.16 (s, 1 F), −111-93 (s, 1 F); HRMS (ESI) m/z calcd forC₁₅H₈Br₂F₄N₂O₂H⁺: 482.8961, Found: 482.8958 (Δ=0.69 ppm).

N′-(3,5-dibromo-2-hydroxybenzylidene) quinolinylhydrazide (A10)

Beige solid (99 S yield); m.p.>215° C.; ³H NMR (700 MHz DMSO-d₆) δ 7.33(t, 1H, J=7.9 Hz), 7.85 (dd, 2H, J=13.4, 2.4 Hz), 7.91 (t, 1H, J=7.7Hz), 8.12 (d, 1H, J=8.5 Hz), 8.16 (d, 1H, J=7.8 Hz), 8.57 (s, 1H), 8.95(s, 1H), 9.34 (s, 1H), 12.63 (s, 1H), 12.85 (s, 1H); ¹³C NMR (175 MHzDMSO-d₆) δ 110.50, 111.33, 120.96, 125.11, 126.36, 127.72, 128.86,129.29, 131.80, 132.19, 135.77, 136.54, 147.50, 148.79, 153.70, 161.75.

4-Cyano-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A11)

White solid (47% yield); m.p.>215° C.; ¹H NMR (500 MHz DMSO-d₆) δ 7.83(s, 2H), 8.05 (d, 2H, J=8.6 Hz), 8.09 (d, 2H, J=8.6 Hz), 8.53 (s, 1H),12.64 (s, 2H); ¹³C NMR (125 MHz DMSO-d₆) δ 110.47, 111.32, 114.50,118.17, 120.90, 128.62, 132.20, 132.65, 135.82, 136.20, 147.89, 153.70,161.76; HRMS (ESI) m/z calcd for C₁₉H₉Br₂N₃O₂H⁺: 421.9134, Found:421.915 (Δ=−3.67 ppm).

2-Fluoro-4-trifluoromethoxy-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide(A12)

Yellow solid (57% yield); m.p. 151-152° C.; ¹H NMR (500 MHz DMSO-d₆) δ6.62 (d, 1H, 30%, J=8.8 Hz), 6.89 (d, 1H, 73%, J=8.8 Hz), 7.31-7.33 (m,2H, 52%), 7.35 (d, 1H, 30%, J=8.8 Hz), 7.38-7.40 (m, 1H, 70%), 7.43 (dd,1H, 67%, J=8.8, 2.6 Hz), 7.52-7.54 (m, 1H, 30%), 7.57-7.60 (m, 1H, 70%),7.67 (t, 1H, 30%, J=8 Hz), 7.80 (s, 1H, 70%), 7.84 (t, 1H, 70%, J=8.2Hz), 8.27 (s, 1H, 28%), 8.49 (s, 1H, 72%), 10.27 (s, 1H, 30%), 11.03 (s,1H, 70%), 12.15 (s, 1H, 35%), 12.19 (s, 1H, 65%); ¹³C NMR (125 MHzDMSO-d₆) δ 109.89, 110.10, 110.53, 117.17, 118.68, 121.25, 121.99,122.01, 122.13, 128.17, 130.08, 131.01, 131.05, 131.92, 131.95, 133.45,133.89, 140.53, 145.79, 150.32, 150.41, 155.60, 156.38, 158.48, 159.29,160.49, 165.78; ¹⁹F NMR (376 MHz DMSO-d₆) δ −56.95 (s, 3 F), −57.04 (s,3 F), −109.09 (s, 1 F), 109.47 (s, 1 F); HRMS (ESI) m/z calcd forC₁₅H₉BrF₄N₂O₃H⁺: 420.9805, Found: 420.982 (Δ=−3.47 ppm).

2-Fluoro-4-trifluoromethoxy-N′-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide(A13)

Yellow solid (62% yield); m.p. 206-207° C.; ¹H NMR (500 MHz DMSO-d_(F))δ 7.41 (d, 1H, J=8.6 Hz), 7.61 (d, 1H, J=8.8 Hz), 7.83 (dd, 2H, J=7.3,2.4 Hz), 7.87 (t, 1H, J=8.3 Hz), 8.45 (s, 1H), 12.39 (s, 1H), 12.57 (s,1H); ¹³C NMR (125 MHz DMSO-d₆) δ 109.92, 110.13, 110.52, 111.10, 111.32,111.84, 117.18, 117.42, 118.77, 120.88, 121.31, 121.43, 122.21, 122.88,130.60, 130.93, 132.07, 132.10, 132.19, 135.60, 135.87, 143.31, 147.78,150.58, 150.67, 153.62, 158.57, 159.41, 160.59; 165.56; ¹⁹F NMR (376 MHzDMSO-d₆) δ −56.94 (s, 3 F), −57.02 (s, 3 F), −109.09 (s, 1 F), −109.94(s, 1 F); HRMS (ESI) m/z calcd for C₁₅H₈Br₂F₄N₂O₃H⁺: 498.8911, Found:498.8923 (Δ=−2.57 ppm).

3-Bromo-N′-(2-hydroxy-5-trifluoromethylbenzylidene)benzohydrazide (A14)

White solid (92% yield); m.p. 175-176° C.; ¹H NMR (400 MHz DMSO-d₆) δ7.10 (d, 1H, 90%, J=8.6 Hz), 7.15 (d, 1H, 10%, J=8.5 Hz), 7.51 (t, 1H,100%, J=7.9 Hz), 7.62 (dd, 1H, 90%, J=8.6, 1.5 Hz), 7.73 (m, 1H, 10%),7.81 (d, 1H, 100%, J=7.8 Hz), 7.93 (d, 1H, 100%, J=7.9 Hz), 8.00 (s, 1H,95%), 8.09 (s, 1H, 5%), 8.12 (s, 1H, 100%), 8.71 (s, 1H, 90%), 9.06 (s,1H, 10%), 11.64 (s, 1H, 10%), 11.71 (s, 1H, 90%), 12.27 (s, 1H, 90%);¹³C NMR (100 MHz DMSO-d₆) δ 117.19, 119.69, 119.92, 120.24, 120.57,121.77, 123.06, 125.24, 125.75, 126.93, 128.02, 130.18, 130.82, 134.74,134.94, 145.73, 160.00, 161.52; ¹⁹F NMR (376 MHz DMSO-d₆) δ −56.92 (s, 3F), −60.12 (s, 3 F); HRMS (ESI) m/z calcd for C₁₅H₁₀BrF₃N₂O₂H⁺:386.9951, Found: 386.9957 (Δ=−1.78 ppm).

4-Methoxymethyl-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A5)

Yellow solid (84% yield); m.p.>215° C.; ¹H NMR (500 MHz DMSO-d₆) δ 3.31(s, 1H), 3.32 (s, 2H), 4.49 (s, 2H), 7.48 (d, 2H, J=8.1 Hz), 7.81 (d,2H, J=5.3 Hz), 7.94 (d, 2H, J=8.1 Hz), 8.52 (s, 1H), 12.52 (s, 1H),12.74 (s, 1H); ¹³C NMR (125 MHz DMSO-d₆) δ 57.77, 72.95, 110.36, 111.19,120.96, 127.25, 127.82, 131.07, 132.11, 135.52, 142.97, 146.97, 153.65,162.75; HRMS (ESI) m/z calcd for C₁₆H₁₄Br₂N₂O₃H⁺: 440.9444, Found:440.9448 (Δ=−0.88 ppm).

4-Dimethylamino-N′-(4-dibromo-2-hydroxybenzylidene)benzohydrazide (A16)

Yellow solid (93% yield); m.p. 195-196° C.; ¹H NMR (400 MHz DMSO-d₆) δ2.99 (s, 6H), 6.75 (d, 2H, J=9 Hz), 7.09 (dd, 1H, J=8.24, 1.8 Hz), 7.12(s, 1 h), 7.47 (d, 1H, J=8.3 Hz), 7.81 (d, 2H, J=9 Hz), 8.55 (s, 1H),11.76 (s, 1H), 11.85 (s, 1H); ¹³C NMR (100 MHz DMSO-d₆) δ 110.84,118.55, 119.04, 122.27, 123.38, 129.14, 130.65, 145.41, 152.62, 158.03,162.57; HRMS (ESI) m/z calcd for C₁₆H₁₆BrN₃O₂H⁺: 362.0499, Found:362.0504 (o=−1.37 ppm).

3,5-Dibromo-2-hydroxy-N′-(5-methyl-2-hydroxyphenylmethylidene)benzohydrazide(A17)

Yellow solid (88 Yield); m.p 148-153° C.; ¹H NMR (700 MHz DMSO-d₆) δ2.23 (s, 3H), 6.83 (d, 1H, J=8.3 Hz), 7.12 (dd, 1H, J=8.3, 1.9 Hz), 7.43(s, 1H), 8.01 (s, 1H), 8.19 (s, 1H), 8.67 (s, 1H), 10.63 (s, 1H), 12.37(s, 1H), 13.09 (s, 1H); ¹³C NMR (175 MHz DMSO-d₆) δ 19.96, 109.81,112.41, 116.32, 116.57, 118.35, 128.08, 128.48, 129.19, 132.84, 138.74,149.52, 155.39, 156.82, 164.17; MS (ESI) m/z 426.9 (M+1)⁺

3,5-Dibromo-2-hydroxy-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide(A18)

Yellow solid (95: Yield); m.p>230° C.; ¹H NMR (700 MHz DMSO-d₆) δ 7.10(dd, 1H, J=8.3, 1.8 Hz), 7.12 (s, 1H), 7.62 (d, 1H, J=8.3 Hz), 8.00 (s,1H), 8.17 (s, 1H), 8.68 (s, 1H), 11.17 (s, 1H), 12.41 (s, 1H); ¹³C NMR(175 MHz DMSO-d₆) δ 109.78, 112.46, 116.61, 118.59, 119.07, 122.61,124.62, 129.22, 129.54, 138.76, 147.78, 156.83, 158.06, 164.24; MS (ESI)m/z 488.7 (M−1)⁻

3,5-Difluoro-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A19)

White solid (87% yield); m.p. 222-228° C.; ¹H NMR (400 MHz DMSO-d₆) δ6.97 (d, 1H, 25%, J=8.3 Hz), 7.03 (s, 1H, 25%), 7.11 (d, 1H, 75%, 8.3Hz), 7.19 (d, 1H, 25%, J=8.4 Hz), 7.30-7.37 (m, 2H, 252, 100%),7.48-7.51 (m, 1H, 75%), 7.58 (d, 1H, 75%, J=8.3 Hz), 7.60-7.66 (m, 2H,1002, 75%), 8.30 (s, 1H, 26%), 8.52 (s, 1H, 74%), 10.41 (s, 1H, 30%),11.22 (s, 1H, 70%), 12.16 (s, 1H, 100%); ¹³C NMR (100 MHz DMSO-d₆) S118.55, 118.80, 118.88, 119.05, 119.17, 119.87, 120.04, 122.52, 123.69,124.23, 124.59, 124.81, 124.93, 125.27, 125.31, 127.94, 129.95, 141.78,145.94, 146.08, 148.39, 148.52, 148.58, 150.97, 157.18, 157.96, 159.07,165.38; ¹⁹F NMR (376 MHz DMSO-d₆) δ −137.95 (d, 1 F, J=23 Hz), −138.74(d, 1 F, J=23 Hz), −139.00 (d, 1 F, 23 Hz), −139.94 (d, 1 F, J=23 Hz);MS (ESI) m/z 352.9 (M−1)⁻

3,5-Difluoro-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide (A20)

Brown solid (56% yield); m.p. 171-173° C.; ¹H NMR (400 MHz DMSO-d₆) δ6.85 (D, 1H, 25%, J=8.6 Hz), 6.94 (D, 1H, 75%, J=8.8 Hz), 7.20 (S, 1H,15%), 7.21 (s, 1H, 40%), 7.30-7.37 (m, 3H, 100%, 25%, 60%), 7.48-7.52(m, 1H, 75%), 7.55-7.58 (m, 1H, 15%), 7.60-7.64 (m, 1H, 85%), 7.67 (s,1H, 85%), 8.29 (s, 1H, 25%), 8.51 (s, 1H, 75%), 10.25 (s, 1H, 20%),11.01 (s, 1H, 80%), 12.22 (s, 1H, 100%); ¹³C NMR (100 MHz DMSO-d₆) δ118.06, 118.24, 118.94, 119.12, 119.91, 120.08, 121.35, 123.03, 123.07,124.64, 124.79, 124.90, 125.28, 125.38, 127.20, 130.68, 131.11, 141.07,146.05, 155.25, 155.99, 159.16, 159.18; ¹⁹F NMR (376 MHz DMSO-d₆) δ−137.95 (d, 1 F, J=23 Hz), −138.73 (d, 1 F, J=23 Hz), −139.07 (d, 1 F,J=23 Hz), −139.92 (d, 1 F, J=23 Hz); MS (ESI) m/z 309.0 (M−1)⁻

4-Difluoromethoxy-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A21)

Yellow solid (96% yield); m.p. 195-200° C.; ¹H NMR (400 MHz DMSO-d₆) δ7.10-7.14 (m, 2H, 100%), 7.20 (s, 1H, 25%), 7.33 (d, 2H, J=8.5 Hz),7.39, (s, 1H, 50%), 7.56 (d, 2H, 25%, 100%, J=7.4 Hz), 8.01 (d, 2H, 100%J=8.5 Hz), 8.62 (s, 1H, 100%), 11.49 (s, 1H, 100%), 12.14 (s, 1H, 100%);¹³C NMR (100 MHz, DMSO-d₆) δ 113.46, 116.02, 118.07, 118.56, 118.59,119.07, 122.43, 123.92, 129.31, 129.86, 130.31, 146.51, 158.03, 161.84;¹⁹F NMR (376 MHz DMSO-d₀) δ −82.94 (s, 1 F); HRMS (ESI) m/z calcd forC₁₅H₁₁BrF₂N₂O₃H⁺: 384.9994, Found: 385.0007 (Δ=−3.49 ppm).

4-Difluoromethoxy-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (A22)

Beige solid (94% yield); m.p. 194-196° C.; ¹H NMR (400 MHz DMSO-d₆) δ6.90 (d, 1H, 100%, J=8.8 Hz), 7.20 (s, 1H, 25%), 7.33 (d, 2H, 100%,J=8.6), 7.39 (s, 1H, 50%), 7.43 (dd, 1H, 100%, J=8.8, 2.2 Hz), 7.57 (s,1H, 25%), 7.80 (s, 1H, 100%), 8.01 (d, 2H, 100%, J=8.6 Hz), 8.61 (s, 1H,100%), 11.26 (s, 1H, 100%), 12.19 (s, 1H, 100%); ¹³C NMR (100 MHz,DMSO-d₆) δ 110.45, 113.46, 116.03, 118.07, 118.60, 118.68, 121.32,129.28, 129.90, 130.36, 133.60, 145.62, 153.68, 156.41, 161.94; ¹⁹F NMR(376 MHz DMSO-d_(F)) δ −82.94 (s, 1 F); HRMS (ESI) m/z calcd forC₁₅H₁₁BrF₂NO₃H⁺: 384.9994, Found: 385.0019 (Δ=−6.65 ppm).

4-Difluoromethoxy-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide(A23)

Beige solid (89% yield); m.p.>220° C.; ¹H NMR (400 MHz DMSO-d₆) δ 7.21(s, 1H, 25%), 7.35 (d, 2H, 100%, J=8.6 Hz), 7.40 (s, 1H, 50%), 7.58 (s,1H, 25%), 7.83 (s, 2H, 100%), 8.03 (d, 2H, 100% J=8.7 Hz), 8.53 (s, 1H,100%), 12.56 (s, 1H, 100%), 12.70 (s, 1H, 100%); ¹³C NMR (100 MHz,DMSO-d₆) δ 110.38, 111.21, 113.42, 115.99, 118.09, 120.95, 128.65,130.04, 132.12, 135.58, 147.09, 153.66, 162.02; ¹⁹F NMR (376 MHzDMSO-d₆) δ −83.03 (s, 1 F); HRMS (ESI) m/z calcd for C₁₅H₁₀Br₂F₂N₂O₃H⁺:462.9099, Found: 462.9103 (Δ=−0.92 ppm).

3-Trifluoromethyl-N′-(3-chloro-2-hydroxybenzylidene)benzohydrazide (A24)

White solid (68% yield); 3H NMR (500 MHz, DMSO-d₆) δ 6.98 (t, J=7.8 Hz,1H), 7.51 (m, 1H), 7.82 (t, J=7.8 Hz, 1H), 8.01 (d, J=7.8 Hz, 1H), 8.26(d, J=7.9 Hz, 1H), 8.29 (s, 1H), 12.28 (s, 1H), 12.55 (s, 1H). ¹³C NMR(125 MHz, DMSO-d₆) δ 119.6, 120.1, 120.4, 124.2, 128.7, 129.2, 129.5,130.0, 131.5, 131.9, 133.3, 149.2, 153.3, 161.5. ¹⁹F NMR (376 MHz,DMSO-de) δ −61.14 (s, 3H). MP>220° C. HRMS [M+H]⁺ calcd forC₁₁H₁₁ClF₃N₂O₂ ⁺: 343.0456, found: 343.0459 (Δ=−0.9 ppm).

3-Trifluoromethyl-N′-(3-bromo-2-hydroxybenzylidene)benzohydrazide (A25)

White solid (72% yield); 1H NMR (400 MHz, DMSO-d₆) δ 6.93 (t, J=7.8 Hz,1H), 7.55 (dd, J=7.7 Hz, J=1.4 Hz, 1H), 7.65 (dd, J=7.9 Hz, J=1.4 Hz,1H), 7.82 (t, J=7.9 Hz, 1H), 8.02 (d, J=7.9 Hz, 1H), 8.26 (d, J=8.0 Hz,1H), 8.29 (s, 1H), 8.60 (s, 1H), 12.49 (br s, 1H), 12.55 (br s, 1H). ¹³CNMR (125 MHz, DMSO-d₆) δ 110.0, 119.3, 120.6, 124.3, 125.3, 128.8,130.0, 130.4, 132.0, 133.3, 134.5, 149.3, 154.2, 161.5. ¹⁹F NMR (376MHz, DMSO-d₆) δ −61.14 (s, 3H). MP>220° C. HRMS [M+H]⁺ calcd forC₁₅H₁₁BrF₃N₂O₂ ⁺: 386.9951, found: 386.9946 (Δ=1.3 ppm).

3-Trifluoromethyl-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A26)

White solid (73% yield); ¹H NMR (400 MHz, DMSO-d₆) δ 7.13 (m, 2H), 7.60(d, J=8.3 Hz, 1H), 7.80 (t, J=7.7 Hz, 1H), 7.99 (d, J=7.7 Hz, 1H), 8.24(d, J=8.0 Hz, 1H), 8.28 (s, 1H), 8.66 (s, 1H), 11.37 (s, 1H), 12.28 (s,1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 118.6, 119.1, 122.5, 124.1, 128.5,129.1, 129.9, 130.0, 131.9, 133.7, 146.7, 158.0, 161.4. ¹⁹F NMR (376MHz, DMSO-d₆) δ −61.12 (s, 3H). MP>220° C. HRMS [M+H]⁺ calcd forC₁₅H₁₁BrF₃N₂O₂ ⁺: 386.9951, found: 386.9957 (Δ=−1.6 ppm).

3-Fluoro-N′-(3-bromo-2-hydroxybenzylidene)benzohydrazide (A27)

White solid (73% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 6.92 (t, 1H, 75%,J=7.8 Hz), 6.99 (t, 1H, 25%, J=7.8 Hz), 7.50 (td, 1H, 75%, J=8.5 Hz,J=2.2 Hz), 7.54 (d, 1H, 75%, J=7.6 Hz), 7.61-7.65 (m, 2H, 75%, 100%),7.75-7.77 (m, 2H, 25%, 75%), 7.81 (d, 1H, 75%, J=7.7 Hz), 8.58 (s, 1H,75%), 9.13 (s, 1H, 25%), 12.08 (br s, 1H, 25%), 12.45 (br s, 1H, 75%),12.50 (s, 1H, 1002). ¹³C NMR (175 MHz, DMSO-d₆) δ 110.1, 110.1, 114.5,114.6, 118.8, 119.1, 119.3, 119.3, 120.6, 121.1, 124.0, 124.0, 130.5,130.9, 132.1, 134.5, 134.6, 134.7, 136.4, 149.1, 154.2, 155.3, 161.3,161.6, 162.7, 165.1. ¹⁹F NMR (376 MHz, DMSO-d₆) δ −112.26 (td, J=5.6 Hz,J=9.2 Hz, 1F). MP=192-193° C. HRMS [M+H]⁺ calcd for C₁₄H₁₁BrFN₂O₂⁺:386.9982, found: 386.9986 (o=−0.9 ppm).

3-Fluoro-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A28)

White solid (61% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 7.12 (d, 1H, 78%,J=7.8 Hz), 7.14-7.17 (m, 2H, 78%, 24%), 7.19 (s, 1H, 19%), 7.47 (td, 1H,79%, J=2.0 Hz, J=8.5 Hz), 7.58-7.62 (m, 2H, 100%, 78%), 7.67 (d, 1H,20%, J=8.3 Hz), 7.74 (d, 1H, 79%, J=9.7 Hz), 7.79 (d, 1H, 79%, J=7.7Hz), 8.63 (s, 1H, 81%), 9.0 (s, 1H, 21%), 11.42 (s, 1H, 100%), 12.17 (s,1H, 100). ¹³C NMR (175 MHz, DMSO-d₆) δ 114.4, 114.5, 118.0, 118.6,118.9, 119.0, 119.1, 119.3, 122.5, 121.8, 123.9, 123.9, 124.1, 126.1,130.2, 130.8, 131.5, 135.1, 135.1, 136.7, 158.0, 159.2, 161.3, 161.3,161.6, 162.6. ¹⁹F NMR (376 MHz, DMSO-d₆) δ −112.41 (td, J=6.0 Hz, J=9.4Hz, 1F). MP=201-202° C. HRMS [M+H]⁺ calcd for C₄H₁₁BrFN₂O₂ ⁺:386.9982,found: 386.9985 (Δ=−0.8 ppm).

3,4-Dibromo-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A29)

Yellow solid (78% yield); m.p. 225-230° C.; ¹H NMR (400 MHz, DMSO-d₆) δ7.81-7.85 (m, 3H, 100%), 7.93-7.95 (m, 2H, 100%, 20%), 8.27 (s, 1H,80%), 8.50 (s, 1H, 80%), 9.03 (s, 1H, 20%), 12.53 (s, 2H, 100%, 50%);¹³C NMR (100 MHz, DMSO-d6) δ 110.48, 110.76, 111.30, 111.68, 120.37,120.91, 124.28, 128.58, 132.53, 133.45, 135.77, 137.77, 147.64, 153.65,154.75, 160.86, 163.95; HRMS (ESI) m/z calcd for C₁₄H₈Br₄N₂O₂: 552.7391,Found: 552.7392 (Δ=0.18 ppm).

3,4-Dibromo-N′-(3,5-dichloro-2-hydroxybenzylidene)benzohydrazide (A30)

Yellow solid (64% yield); m.p. 210-215° C.; ¹H NMR (400 MHz, DMSO-d₆) δ7.63 (d, 1H) 7.69 (d, 1H) 7.84 (dd, 1H) 7.97 (d, 1H) 8.29 (s, 1H) 8.55(s, 1H) 12.35 (d, 2H) ¹³C NMR (125 MHz, DMSO-d6) δ 120.77, 121.99,123.03, 124.29, 128.36, 128.58, 130.47, 133.05, 134.16, 147.48, 152.24,160.86, 163.54; HRMS (ESI) m/z calcd for C₁₄H₈Br₂Cl₂N₂O₂: 464.8404,Found: 464.8402 (Δ=−0.32 ppm).

3,4-Dibromo-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide (A31)

Light yellow solid (35% yield); m.p.>230° C.; ¹H NMR (400 MHz, DMSO-d₆)δ 6.95 (d, 1H, 60a, J=8.8 Hz), 7.00 (d, 1H, 402%, J=8.8 Hz), 7.32 (dd,1H, 60%, J=8.8, 2.6 Hz), 7.41 (dd, 1H, J=8.8, 2.6 Hz), 7.68 (d, 1H, 60%,J=2.5 Hz), 7.77 (d, 1H, 40%, J=2.6 Hz), 7.85 (dd, 1H, 65%, J=8.3, 1.7Hz), 7.95 (d, 1H, 60%, J=8.3 Hz), 8.29 (s, 1H, 65%), 8.62 (s, 1H, 60%),8.94 (s, 1H, 40%), 11.13 (s, 1H, 100%), 12.25 (s, 1H, 60%); ¹³C NMR (125MHz, DMSO-d₆) δ 118.21, 118.47, 119.94, 120.70, 123.11, 124.19, 127.22,128.01, 128.51, 131.00, 132.44, 133.59, 134.06, 146.06, 155.98, 157.24,160.83; HRMS (ESI) m/z calcd for C₁₄H₉Br₂ClN₂O₂: 430.8812, Found:430.8792 (Δ=−4.51 ppm).

3,4-Dibromo-N′-(2-hydroxy-1-naphthylidene)benzohydrazide (A32)

Dark yellow solid (66% yield); m.p.>230° C.; 1H NMR (400 MHz, DMSO-d₆) δ7.22 (d, 1H, 85?, J=9 Hz), 7.7.27 (d, 1H, 152%, J=9 Hz), 7.40 (t, 1H,100%, J=7.5 Hz), 7.60 (t, 1H, 100%, J=7.6 Hz), 7.87-7.89 (m, 2H, 85%,85%), 7.93 (d, 1H, 100%, J=9 Hz), 7.98 (d, 1H, 85%, J=8.3 Hz), 8.02 (d,1H, 15%, J=8.3 Hz), 8.28 (d, 1H, 85%, J=8.6 Hz), 8.32 (s, 1H, 85%), 8.63(d, 1H, 15%, J=8.6 Hz), 9.44 (s, 1H, 85%), 9.97 (s, 1H, 15%), 12.28 (s,1H, 85%), 12.55 (s, 1H, 85%), 12.86 (s, 1H, 10%); ¹³C NMR (100 MHz,DMSO-d₆) δ 108.55, 118.85, 120.91, 123.64, 124.33, 127.89, 128.12,129.01, 131.60, 132.39, 133.05, 134.23, 147.57, 158.12, 160.35; HRMS(ESI) m/z calcd for C₁₀H₁₁Br₂N₂O₂: 446.934, Found: 446.9338 (Δ=−0.46ppm).

3,4-Dibromo-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (A33)

Light yellow, cream solid (53% yield); m.p.>230° C.; ¹H NMR (400 MHz,DMSO-d₆) δ 6.89 (d, 1H, 65%, J=8.8 Hz), 6.94 (d, 1H, 35%, J=8.8 Hz),7.42 (dd, 1H, 65%, J=8.7, 2.3 Hz), 7.52 (dd, 1H, 35%, J=8.8, 2.4 Hz),7.80 (s, 1H, 65%), 7.83 (d, 1H, 70%, J=8.3 Hz), 7.88 (s, 1H, 35%), 7.94(d, 1H, 70%, J=8.3 Hz), 8.28 (s, 1H, 70%), 8.60 (s, 1H, 65%), 8.92 (s,1H, 35%), 11.13 (s, 1H, 100%), 12.23 (s, 1H, 60%); ¹³C NMR (100 MHz,DMSO-d₆) δ 110.38, 118.73, 121.15, 124.02, 128.35, 129.91, 132.28,133.44, 133.61, 145.72, 156.23, 157.48, 160.56; HRMS (ESI) m/z calcd forC₁₄H Br₃N₂O: 474.8288, Found: 474.8287 (Δ=−0.19 ppm).

3,4-Dibromo-N′-(2-hydroxy-5-methylbenzylidene)benzohydrazide (A34)

White solid (94% yield); m.p. 212-216° C.; ¹H NMR (400 MHz DMSO-d₆) δ2.24 (s, 3H), 6.82 (d, 1H, 55%, J=8.3 Hz), 6.86 (d, 1H, J=8.3 Hz), 7.10(d, 1H, 55%, J=8.2 Hz), 7.19 (d, 1H, 45%, J=8.3 Hz), 7.36 (s, 1H, 55%),7.47 (s, 1H, 45%), 7.83 (d, 1H, 60%, J=8.3 Hz), 7.94 (d, 2H, 30%, 30?,J=8.3 Hz), 8.27 (s, 1H, 60%), 8.58 (s, 1H, 55%), 8.91 (s, 1H, 45%),10.84 (s, 1H, 60%), 10.90 (s, 1H, 40%), 12.15 (s, 1H, 50%); ¹³C NMR (100MHz, DMSO-d₆) δ 116.38, 118.36, 124.19, 128.14, 128.49, 132.42, 148.46,155.28, 156.49, 160.54, 162.47; HRMS (ESI) m/z calcd for C₁₅H₁₁Br₂N₂O₂:410.9343, Found: 410.9338 (Δ=−1.25 ppm).

3,4-Dibromo-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A35)

Off-white solid (63% yield); m.p.>230° C.; ¹H NMR (400 MHz, DMSO-d₆) δ7.08-7.18 (m, 2H, 1002, 100%), 7.57 (d, 1H, 80%, J=8.3 Hz), 7.65 (d, 2H,20%, 20%, J=8.3 Hz), 7.83 (dd, 1H, 80%, J=8.3, 1.9 Hz), 7.94 (d, 1H,80%, J=8.3 Hz), 8.27 (s, 1H, 80%), 8.61 (s, 1H, 75%), 8.94 (s, 1H, 25%),11.33 9 s, 1H, 100%), 12.18 (s, 1H, 75%); ¹³C NMR (100 MHz, DMSO-d_(F))δ 118.60, 122.49, 124.11, 126.08, 128.48, 130.02, 132.42, 146.74,157.99, 160.61; HRMS (ESI) m/z calcd for C₁₄H₈Br₄N₂O₂: 474.8286, Found:474.8287 (Δ=0.16 ppm).

3,5-Dibromo-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (A36)

White solid (58 k yield); m.p.>230° C.; ¹H NMR (400 MHz, DMSO-de) δ 6.90(d, 1H, J=8.8 Hz), 7.43 (dd, 1H, J=8.8, 2.6 Hz), 7.82 (d, 1H, J=2.5 Hz),8.11 (s, 3H), 8.61 (s, 1H), 11.10 (s, 1H), 12.25 (s, 1H); ¹³C NMR (100MHz, DMSO-d₆) δ 110.52, 118.67, 121.33, 122.72, 129.60, 129.95, 136.40,136.59, 145.99, 156.39; HRMS (ESI) m/z calcd for C₁₄H₉BrN₂O₂: 474.8288,Found: 474.8287 (Δ=−0.32 ppm).

3,5-Dibromo-N′-(2-hydroxy-1-naphthylidene)benzohydrazide (A37)

Yellow solid (42% yield); m.p.>230° C.; ¹H NMR (700 MHz, DMSO-d₆) δ 7.24(d, 1H, J=8.9 Hz), 7.42 (t, 1H, J=7.4 Hz), 7.62 (t, 1H, J=7.6 Hz), 7.90(d, 1H, J=8 Hz), 7.95 (d, 1H, J=8.9 Hz), 8.14 (s, 1H), 8.16 (s, 2H),8.32 (d, 1H, J=8.6 Hz), 9.44 (s, 1H), 12.31 (s, 1H), 12.50 (s, 1H); ¹³CNMR (175 MHz, DMSO-d₆) δ 108.54, 118.82, 120.99, 122.85, 123.63, 127.86,127.90, 129.00, 129.56, 131.58, 133.12, 136.84, 136.69, 147.79, 158.15,159.66; HRMS (ESI) m/z calcd for C₁₄H₉Br₃N₂O₂: 446.934, Found: 446.9338(Δ=−0.34 ppm).

3,5-Dibromo-N′-(3,5-dichloro-2-hydroxybenzylidene)benzohydrazide (A38)

Tan solid (Quantitative yield); m.p.>230° C.; ¹H NMR (400 MHz, DMSO-d₆)δ 7.63 (s, 1H), 7.69 (s, 2H), 8.11 (s, 2H), 8.12 (s, 1H), 8.56 (s, 1H),12.25 (s, 1H), 12.61 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 120.78,121.62, 122.81, 123.08, 128.34, 129.70, 130.55, 135.85, 136.91, 147.67,152.23, 160.24; HRMS (ESI) m/z calcd for C₁₄H₈Br₂Cl₂NO₂: 446.9339,Found: 446.9338 (Δ=−0.19 ppm).

3,5-Dibromo-N′-(4-bromo-2-hydroxybenzylidene)benzohydrazide (A39)

White solid (84% yield); m.p.>230° C.; ¹H NMR (700 MHz, DMSO-d₆) δ 7.15(d, 1H, J=8.3 Hz), 7.14 (s, 1H), 7.59 (d, 1H, J=8.3 Hz), 8.10 (s, 3H),8.62 (s, 1H), 11.30 (s, 1H), 12.20 (s, 1H); ¹³C NMR (175 MHz, DMSO-d₆)118.64, 119.06, 122.53, 122.76, 124.21, 129.60, 129.89, 136.46, 136.60,146.81, 157.99, 159.98; HRMS (ESI) m/z calcd for C₁₄H₉Br₃N₂O₂: 474.8289,Found: 474.8287 (Δ=−0.42 ppm).

3,5-Dibromo-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide (A40)

White solid (92% yield); m.p.>230° C.; ¹H NMR (700 MHz, DMSO-d₆) δ 6.95(d, 1H, J=8.8 Hz), 7.33 (dd, 1H, J=8.8, 2.7 Hz), 7.69 (s, 1H), 8.11 (s,3H), 8.62 (s, 1H), 11.09 (s, 1H), 12.25 (s, 1H); ¹³C NMR (175 MHz,DMSO-d₆) δ 118.24, 120.74, 122.76, 127.09, 129.62, 131.09, 136.41,146.15, 156.00, 160.08; HRMS (ESI) m/z calcd for C₁₄H₉Br₂C₁N₂O₂:430.8802, Found: 430.8792 (Δ=−2.34 ppm).

3,5-Dibromo-N′-(2-hydroxy-5-methylbenzylidene)benzohydrazide (A41)

White solid (92% yield); m.p.>230° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 2.25(s, 3H), 6.82 (d, 1H, J=8.2 Hz), 7.10 (dd, 1H, J=8.3, 1.8 Hz), 7.38 (s,1H), 8.09 (s, 3H), 8.91 (s, 1H), 10.78 (s, 1H), 12.15 (s, 1H); ¹³C NMR(175 MHz, DMSO-d₆) δ 19.96, 116.29, 116.41, 117.88, 122.76, 128.00,128.87, 129.64, 130.45, 131.22, 132.45, 133.95, 136.55, 148.56, 155.30,156.52, 159.90, 162.50; HRMS (ESI) m/z calcd for C₁₅H₁₂Br₂N₂O₂:410.9345, Found: 410.9338 (Δ=−1.65 ppm).

2,3-Dibromo-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A42)

White solid (98% yield); m.p. 227-230° C.; ¹H NMR (400 MHz, DMSO-d₆) δ7.42-7.46 (m, 2H, 30%, 100%), 7.56-7.58 (m, 2H, 70%, 30%), 7.74 (s, 1H,25%), 7.82 (s, 1H, 75%), 7.84 (s, 1H, 70%), 7.88 (d, 1H, 20%), 7.90 (dd,1H, 80%, J=7.9, 1.4 Hz), 8.21 (s, 1H, 25%), 8.38 (s, 1H, 75%), 10.30 (s,1H, 25%), 12.32 (s, 1H, 75%), 12.47 (s, 1H, 35%), 12.60 (s, 1H, 65%);¹³C NMR (175 MHz, DMSO-d₆) δ 110.57, 111.54, 120.77, 120.93, 121.54,121.83, 125.15, 125.45, 127.18, 128.14, 129.52, 129.74, 131.33, 132.14,134.45, 135.17, 135.68, 135.93, 139.15, 139.70, 144.00, 147.75, 152.57,153.63, 163.06, 168.31; HRMS (ESI) m/z calcd for C₁₁H₈Br₄N₂O₂: 552.7393,Found: 552.7392 (Δ=−0.19 ppm).

2,3-Dibromo-N′-(3,5-dichloro-2-hydroxybenzylidene)benzohydrazide (A43)

Product washed with water and −1 mL ethyl acetate, filtered, and washedwith DCM and hexanes. Tan solid (43% yield); m.p. 227-230° C.; ¹H NMR(500 MHz DMSO-d₆) δ 7.37 (d, 1H, 30%, 2.6 Hz), 7.42-7.46 (m, 3H, 30%,70%, 30%), 7.53 (d, 1H, 30%, J=2.6 Hz), 7.57 (dd, 1H, 70%, J=7.6, 1.5Hz), 7.64 (d, 1H, J=2.6 Hz), 7.68 (d, 1H, 70%, J=2.6 Hz), 7.89 (dd, 1H,40%, J=6.7, 2.9 Hz), 7.91 (dd, 1H, 60%, J=8, 1.5 Hz), 8.25 (s, 1H, 30%),8.42 (s, 1H, 70%), 10.27 (s, 1H, 30%), 12.08 (s, 1H, 70%), 12.45 (s, 1H,30%), 12.56 (s, 1H, 70%); ¹³C NMR (125 MHz, DMSO-de) δ 120.84, 121.80,123.15, 123.41, 125.03, 127.15, 128.28, 129.51, 130.61, 134.32, 135.07,139.19, 139.87, 143.35, 147.38, 151.07, 152.16, 163.00, 168.35; MS (ESI)m/z 462.8 (M−1)⁻

4-Fluromethyl-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A44)

White solid (46% yield); m.p.>230° C.; ¹H NMR (400 MHz DMSO-d₆) δ 5.46(s, 1H), 5.58 (s, 1H), 7.57 (d, 2H, J=7.6 Hz), 7.80-7.82 (m, 2H), 7.99(d, 2H, J=7.9 Hz), 8.53 (s, 1H), 12.56 (s, 1H), 12.71 (s, 1H); ¹³C NMR(100 MHz, DMSO-d₆) δ 82.71, 84.33, 110.39, 111.21, 120.95, 127.27,127.33, 128.05, 132.13, 135.58, 140.40, 140.57, 147.17, 153.66, 162.61;¹⁹F NMR (376 MHz DMSO-d₆) δ −209.61 (t, 1 F, J=47.2 Hz); MS (ESI) m/z428.9 (M+1)⁺

4-Azido-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A45)

White solid (640 yield); m.p. 193-197° C.; ¹H NMR (400 MHz DMSO-d₆) δ7.28 (d, 2H, J=8.6 Hz), 7.79-7.81 (m, 2H), 8.00 (d, 2H, J=8.6 Hz), 8.51(s, 1H), 12.53 (s, 1H), 12.71 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ110.39, 111.21, 119.26, 120.98, 128.45, 129.74, 132.11, 135.55, 143.53,146.99, 153.65, 162.05.

4-Ethynyl-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A46)

Yellow solid (55% yield); m.p.>230° C.; ¹H NMR (300 MHz DMSO-d₆) δ 4.46(s, 1H), 7.39 (d, 1H, J=7.9 Hz), 7.65 (d, 1H, J=8.4 Hz), 7.80-7.96 (m,4H), 8.52 (s, 1H), 12.48-12.66 (m, 2H); MS (ESI) m/z 418.9 (M−1)⁻

4-Ethynyl-N′-(3,5-dibromo-2-hydroxybenzylidene)benzohydrazide (A47)

Yellow solid (75% yield); m.p.>230° C.; ¹H NMR (300 MHz DMSO-d₆) δ 4.44(s, 1H), 6.89 (d, 1H, J=8.7 Hz), 7.35-7.44 (m, 2H), 7.63 (d, 1H, J=8Hz), 7.78-7.95 (m, 2H), 8.60 (s, 1H), 11.23-11.32) m, 1H), 12.13-12.24(m, 1H); MS (ESI) m/z 340.9 (M−1)⁻

Chemical Synthesis and Characterization of Heteroaromatic Acylhydrazonesof this Invention

5-Bromo-N′-(3,5-dibromo-2-hydroxybenzylidene)pyrimidine-2-carbohydrazide(H1)

To a solution of 5-bromopyrimidine-2-carbohydrazide (50 mg, 0.23 mmol),3,5-dibromo-2-hydroxybenzaldehyde (67 mg, 0.24 mmol) in methanol (5 mL)was added 1 drop of glacial acetic acid. The reaction mixture wasstirred at room temperature overnight. Addition of water to the reactionmixture resulted in precipitation of the product, which was filtered,washed with water and dried under vacuum, to give pure product as whitesolid (95 mg, 86% yield: ¹H NMR (700 MHz, DMSO-d₆) δ 7.73 (d, J=8.3, 2.2Hz, 1H), 7.84 (t, J=3.1 Hz, 1H), 8.73 (s, 1H), 9.23 (s, 2H), 12.63 (brs, 1H), 13.06 (s, 1H). ¹³C NMR (175 MHz, DMSO-d₆) δ 110.5, 110.7, 111.3,111.7, 120.4, 120.9, 122.9, 132.2, 133.4, 135.9, 137.8, 149.0, 153.8,154.8, 154.9, 158.4, 158.5, 164.0. MP>220° C. HRMS [M+H]⁺ calcd forC₁H₈Br₃N₄O₂ ⁺ 476.8192, found 476.8183 (Δ=1.9 ppm).

The procedure detailed in the example above can be used for thesynthesis of the following compounds.

5-Bromo-N′-(5-bromo-2-hydroxybenzylidene)thiophene-2-carbohydrazide (H2)

White solid (85% yield); ¹H NMR (500 MHz, DMSO-d₆) δ 6.90 (d, J=8.8 Hz,1H), 7.43 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 7.69 (d, J=1.1 Hz, 1H), 7.80 (d,J=2.4 Hz, 1H), 8.31 (d, J=1.1 Hz, 1H), 8.57 (s, 1H), 11.15 (s, 1H),12.01 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 110.5, 112.5, 118.6, 121.4,129.4, 130.1, 132.6, 133.6, 135.8, 145.2, 156.3, 157.2. MP: 210-211° C.HRMS [M+H]⁺ calcd for C₁₂H₉Br₂N₂O₂S⁺ 402.8746, found 402.8755 (Δ=−2.1ppm).

5-Bromo-N′-(4-bromo-2-hydroxybenzylidene)thiophene-2-carbohydrazide (H3)

White solid (89% yield); ¹H NMR (400 MHz, DMSO-d₆) δ 7.11 (m, 2H), 7.57(d, J=8.2 Hz, 1H), 7.68 (d, J=1.2 Hz, 1H), 8.30 (d, J=1.2 Hz, 1H), 8.58(s, 1H), 11.35 (s, 1H), 11.95 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ112.5, 118.6, 119.0, 122.4, 123.9, 129.4, 130.0, 132.5, 135.8, 146.1,157.2, 157.9. MP: 217-219° C. HRMS [M+H]⁺ calcd for C₁₂H₉Br₂N₂O₂S⁺402.8746, found 402.8757 (Δ=−2.9 ppm).

5-Bromo-N′-(3,5-dibromo-2-hydroxybenzylidene)thiophene-2-carbohydrazide

White solid (91% yield); ¹H NMR (500 MHz, DMSO-d₆) δ 7.69 (d, J=1.2 Hz,1H), 7.83 (s, 2H), 8.34 (d, J=1.4 Hz, 1H), 8.48 (s, 1H), 12.40 (s, 1H),12.56 (s, 1H). 13C NMR (125 MHz, DMSO-d₆) δ 110.4, 111.2, 112.8, 121.0,129.4, 132.1, 133.2, 135.1, 135.6, 146.9, 153.6, 157.3. MP>220° C. HRMS[M+H]⁺ calcd for C₁₂H₈Br₃N₂O₂S⁺ 480.7851, found 480.7856 (Δ=−1.0 ppm).

5-Bromo-N′-((2-hydroxynaphthalen-1-yl)methylene)thiophene-2-carbohydrazide(H5)

White solid (78% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 7.23 (d, J=8.9 Hz,1H), 7.41 (t, J=7.4 Hz, 1H), 7.61 (t, J=7.5 Hz, 1H), 7.71 (d, J=1.5 Hz,1H), 7.90 (d, J=8.0 Hz, 1H), 7.94 (d, J=9.0 Hz, 1H), 8.28 (d, J=8.6 Hz,1H), 8.33 (d, J=1.4 Hz, 1H), 9.40 (s, 1H), 12.07 (s, 1H), 12.57 (s, 1H).¹³C NMR (125 MHz, DMSO-d₆) δ 108.6, 112.8, 118.9, 120.9, 123.6, 127.9,129.0, 129.3, 131.6, 132.6, 132.9, 135.7, 146.7, 156.9, 158.0. MP:197-198° C. HRMS [M+H]⁺ calcd for C₁₆H₁₂BrN₂O₂S⁺ 374.9797, found374.9809 (Δ=−3.2 ppm).

5-Bromo-N′-(4-bromo-2-hydroxybenzylidene)nicotinohydrazide (H6)

White solid (83% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 7.12 (dd, J=8.3 Hz,J=1.8 Hz, 1H), 7.14 (d, J=1.8 Hz, 1H), 7.61 (d, J=8.3 Hz, 1H), 8.51 (t,J=2.1 Hz, 1H), 8.62 (s, 1H), 8.92 (d, J=2.2 Hz, 1H), 9.04 (d, J=1.8 Hz,1H), 11.28 (s, 1H), 12.27 (s, 1H). ¹³C NMR (175 MHz, DMSO-d₆) δ 118.6,119.1, 120.1, 122.5, 124.3, 129.8, 130.3, 137.7, 146.8, 147.3, 153.1,158.0, 160.0. MP>220° C. HRMS [M+H]⁺ calcd for C₃H₁₀Br₂N₃O₂ ⁺: 397.9134,found 397.9147 (Δ=−3.3 ppm).

4,5-Dibromo-N′-(5-bromo-2-hydroxybenzylidene) furan-2-carbohydrazide(H7)

White solid (67 k yield); 1H NMR (700 MHz, DMSO-d₆) δ 6.89 (d, J=8.8 Hz,1H), 7.43 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.56 (s, 1H), 8.79 (d, J=2.2 Hz,1H), 8.61 (s, 1H), 11.01 (s, 1H), 12.23 (s, 1H). ¹³C NMR (175 MHz,DMSO-d₆) δ 103.6, 110.5, 118.7, 119.2, 121.4, 127.2, 129.8, 133.8,145.8, 148.1, 152.4, 156.3. MP>220° C. HRMS [M+H]⁺ calcd for C₂H₈Br₃N₂O₃464.8080, found 464.8092 (Δ=−2.6 ppm).

4,5-Dibromo-N′-(4-bromo-2-hydroxybenzylidene)furan-2-carbohydrazide (H8)

White solid (73% yield); ¹H NMR (400 MHz, DMSO-d₆) δ 7.10 (dd, J=8.2 Hz,J=1.8 Hz, 1H), 7.12 (d, J=1.7 Hz, 1H), 7.55 (s, 1H), 7.57 (d, J=8.3 Hz,1H), 8.62 (s, 1H), 11.21 (s, 1H), 12.18 (s, 1H). ¹³C NMR (100 MHz,DMSO-d₆) δ 103.6, 118.7, 119.0, 119.1, 122.5, 124.1, 127.0, 129.7,146.6, 148.1, 152.3, 157.9. MP>220° C. HRMS [M+H]⁺ calcd forC₁₂H₈Br₃N₂O₃ ⁺ 464.8080, found 464.8073 (Δ=1.5 ppm).

4,5-Dibromo-N′-(3,5-dibromo-2-hydroxybenzylidene)furan-2-carbohydrazide(H9)

White solid (79% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 7.60 (s, 1H), 7.81(d, J=2.3 Hz, 1H), 7.84 (d, J=2.3 Hz, 1H), 8.53 (s, 1H), 12.38 (s, 1H),12.66 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 103.8, 110.5, 111.4, 119.8,121.0, 127.6, 132.0, 135.8, 147.6, 147.8, 152.4, 153.5. MP>220° C. HRMS[M+H]⁺ calcd for C₁₂H₇Br₄N₂O₃ ⁺ 542.7185, found 542.7190 (Δ=−0.9 ppm).

4,5-Dibromo-N′-(2-hydroxynaphthalen-1-yl)methylenefuran-2-carbohydrazide(H10)

White solid (77% yield); ¹H NMR (400 MHz, DMSO-d₆) δ 7.23 (d, J=9.0 Hz,1H), 7.41 (t, J=7.4 Hz, 1H), 7.59 (s, 1H), 7.61 (t, J=7.3 Hz, 1H), 7.90(d, J=8.0 Hz, 1H), 7.94 (d, J=8.9 Hz, 1H), 8.29 (d, J=8.6 Hz, 1H), 9.48(s, 1H), 12.30 (s, 1H), 12.41 (s, 1H). ¹³C NMR (100 MHz, DMSO-d_(b)) δ103.9, 108.6, 118.8, 119.3, 121.0, 123.6, 127.0, 127.8, 128.9, 131.6,133.1, 147.9, 148.1, 152.0, 158.0. MP>220° C. HRMS [M+H]⁺ calcd forC₁₆H₁₁Br₂N₂O₃ ⁺ 436.9131, found 436.9148 (Δ=−3.9 ppm).

4-Bromo-N′-(5-bromo-2-hydroxybenzylidene)furan-2-carbohydrazide (H11)

White solid (67% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 6.90 (d, J=8.8 Hz,1H), 7.43 (dd, J=8.7 Hz, J=2.1 Hz, 1H), 7.48 (s, 1H), 7.78 (s, 1H), 8.23(s, 1H), 8.61 (s, 1H), 11.05 (s, 1H), 12.21 (s, 1H). ¹³C NMR (175 MHz,DMSO-d₆) δ 100.6, 110.5, 117.3, 118.6, 121.4, 130.0, 133.7, 144.4,145.7, 146.9, 153.1, 156.3. MP: 203-205° C. HRMS [M+H]⁺ calcd forC₁₂H₉Br₂N₂O₃ ⁺ 386.8974, found 386.8977 (Δ=−0.8 ppm).

4-Bromo-N′-(4-bromo-2-hydroxybenzylidene) furan-2-carbohydrazide (H12)

White solid (69% yield); ¹H NMR (500 MHz, DMSO-d₆) δ 7.11 (m, 2H), 7.47(s, 1H), 7.57 (d, J=8.3 Hz, 1H), 8.23 (d, J=0.6 Hz, 1H), 8.62 (s, 1H),11.26 (s, 1H), 12.17 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 100.7, 117.3,118.7, 119.0, 122.5, 124.1, 129.9, 144.4, 146.5, 146.9, 153.1, 157.9.MP>220° C. HRMS [M+H]⁺ calcd for C₁₂H₉Br₂N₂O₃ ⁺ 386.8974, found 386.8975(Δ=−0.2 ppm).

4-bromo-N′-(3,5-dibromo-2-hydroxybenzylidene)furan-2-carbohydrazide(H13)

White solid (72% yield); ¹H NMR (700 MHz, DMSO-d₆) δ 7.52 (s, H), 7.81(d, J=2.0 Hz, 1H), 7.84 (d, J=2.4 Hz, 1H), 8.27 (s, 1H), 8.53 (s, 1H),12.44 (s, 1H), 12.63 (s, 1H). ¹³C NMR (175 MHz, DMSO-d₆) δ 100.9, 110.5,111.3, 118.0, 121.0, 132.1, 135.7, 144.8, 146.5, 147.6, 153.2, 153.6.MP=214-215° C. HRMS [M+H]⁺ calcd for C₁₂H₂Br₃N₂O₃ ⁺ 464.8080, found463.8086 (Δ=−1.3 ppm).

N′-(4-bromo-2-hydroxybenzylidene) furan-3-carbohydrazide (H14)

White solid (82% yield); ¹H NMR (700 MHz, DMSO-d_(F)) δ 7.11 (dd, J=8.2Hz, J=1.6 Hz, 1H), 7.14 (d, J=1.7 Hz, 1H), 7.56 (d, J=8.3 Hz, 1H), 7.60(dd, J=5.0 Hz, J=1.0 Hz, 1H), 7.68 (dd, J=5.0 Hz, J=3.0 Hz, 1H), 8.32(d, J=1.8 Hz, 1H), 8.59 (s, 1H), 11.48 (s, 1H), 11.97 (s, 1H). ¹³C NMR(175 MHz, DMSO-d₆) δ 118.6, 119.0, 122.4, 123.8, 126.8, 127.3, 130.2,135.5, 145.8, 157.9, 158.4. MP>220° C. HRMS [M+H]⁺ calcd forC₁₂H₁₀BrN₂O₃ ⁺ 308.9869, found 308.9871 (Δ=−0.6 ppm).

N′-(3,5-dibromo-2-hydroxybenzylidene)tetrahydro-2H-pyran-4-carbohydrazide(H15)

White solid (67% yield); ³H NMR (500 MHz, DMSO-d₆) δ 1.68 (s, 3H), 2.00(s, 2H), 2.19 (s, 1H), 2.50 (s, 1H), 3.32-3.36 (m, 3H), 3.89 (d, 1H,J=2.2), 7.74-7.79 (m, 2H), 8.16-8.29 (m, 1H), 12.02 (s, 1H), 12.58 (d,1H, J=3.97). ¹³C NMR (125 MHz, DMSO-d₆) δ 21.19, 28.49, 66.21, 111.03,122.90, 132.04, 135.29, 140.97, 145.73, 153.47, 165.79, 171.45. MP>220°C. HRMS [M+H]⁺ calcd for C₁₃H₁₅Br₂N₂O₃ ⁺ 404.9444, found 404.9443 (Δ=0.2ppm).

Example 8: In Vitro Activities (MIC₈₀ and K₁₀₀) of Acylhydrazones

In Vitro Susceptibility (MIC₈₀) Assay

MICs was determined following the methods of the Clinical and LaboratoryStandards Institutes (CLSI) with modifications. Yeast Nitrogen Base(YNB) medium (pH 7.0, 0.2% glucose) buffered with HEPES was used for MICstudies. HEPES was used instead of morpholinepropanesulfonic acid(MOPS), because MOPS was found to inhibit the activity of this kind ofcompounds. The compound was serially diluted from 16 to 0.03 μg/ml, in a96-well plate. The inoculum was prepared as described in the CLSIprotocol M27A3 guidelines. The plates were incubated at 37° C. with 5%CO₂ for 24 to 72 h and the optical density was measure at 450 nm. TheMICs was determined as the lowest concentration of the compound thatinhibited 80% of growth compared to the control.

In Vitro Killing Activity (K₁₀₀) Assay

C. neoformans cells from a culture grown overnight were washed in PBSand resuspended in YNB buffered with HEPES at pH 7.4. The cells werecounted, and 2×10⁴ cells were incubated with different concentration ofcompounds in a final volume of 10 ml with a final concentration of 0.5%DMSO. The tubes were then incubated at 37° C. with 5% CO₂ on a rotaryshaker at 200 rpm. Aliquots were taken at time points and diluted, and100-1 portions were plated onto Yeast Extract-Peptone-Dextrose (YPD)plates. YPD plates were incubated in a 30° C. incubator and after 48 h,the numbers of CFU were counted and recorded.

TABLE 4 MIC₈₀ and killing activity (K₁₀₀) of aromatic acylhycirazones,Compound MIC₈₀ (μg/mL) K₁₀₀* (μg/mL) A1 0.25 >1 A2 1 >4 A3 0.25 2 A41 >4 A5 0.12 Fungistatic A6 0.12 0.5 A7 0.5 >4 A8 0.5 1 A9 0.12 0.5 A100.25 Fungistatic A11 0.06 >1 A12 0.5 0.5 A13 0.06 0.5 A14 0.5 >2 A150.007 0.03 A16 0.25 >1 A17 0.25 0.25 A18 0.25 0.25 A19 0.5 0.25 A20 10.5 A21 0.12 — A22 0.25 — A23 0.12 — A24 1 4 A25 1 2 A26 0.5 1 A27 1 2A28 0.5 >2 A29 0.06 >0.5 A30 <0.03 >0.025 A31 0.06 >0.5 A32 0.12 >1 A330.25 2 A34 0.12 >1 A35 0.06 >5 A36 0.06 >5 A37 0.25 0.5 A38 0.5 0.5 A391 1 A40 0.12 1 A41 0.5 1 A42 0.5 0.5 A43 1 1 A44 0.5 0.5 A45 0.06 — A460.12 Fungistatic A47 0.12 0.12 *The minimum concentration of a compoundthat kills 100% of C. neoformans cells in 48 h.

TABLE 5 MIC₈₀ and killing activity (K₁₀₀) of heteroaromaticacylhydrazones. Compound MIC₈₀ (μg/mL) K₁₀₀* (μg/mL) H1 0.25 >2 H20.12 >0.5 H3 0.12 >0.5 H4 0.06 >0.25 H5 0.12 >0.5 H6 1 >4 H7 0.25 >1 H80.06 >0.25 H9 0.12 >0.5 H10 0.12 >0.5 H11 0.25 >1 H12 0.5 >2 H130.06 >0.25 H14 0.5 >1 H15 0.5 >2 *The minimum concentration of acompound that kills 100% of C. neoformans cells in 48 h.

Example 9: In Vivo Efficacy Evaluation (Survival Study) of Compound A15in an Animal Model

For survival study, 4-week old CBA/J (Envigo) male mice were used. Theywere divided as ten mice for each treatment or control group. Mice wereinfected intranasally with 20 μl of a suspension containing 5×10⁵ C.neoformans cells and subsequently treated orally with 20 mg/kg/day ofcompound A15 and fluconazole as drug control in a final volume of 100 μlof PEG30% in a saline buffer. The untreated control group mice received100 μl of PEG30% in a saline buffer. Gavage was used as route ofadministration. At the end of the experiment, 80% of the mice treatedwith compound A15 survived whereas, only 30% of the mice treated withfluconazole survived. Mice were fed ad libitum and monitored every dayfor discomfort and meningitis signs. Mice showing weight loss, lethargy,tremor or inability to reach food or water were sacrificed and survivalwas counted until that day (see FIG. 2).

DISCUSSION

The compounds described herein potent killing activity with low or notoxicity that can be used alone or in combination of current antifungalagents to treat superficial or invasive fungal infections.

A particular challenge with the discovery of antifungal drugs istoxicity due to the similarities between the fungal and human eukaryoticgenomes. In exploring potential therapeutic targets, it became apparentthat fungal sphingolipid pathways are quite distinct from humansphingolipid pathways. In addition, it is well established that thesphingolipid pathway is involved in the virulence of clinicallyimportant pathogenic fungi including Cryptococcus neoformans (Cn). Workfrom our lab and others showed that fungal sphingolipid complex,glucosylceramide (GlcCer), has increased expression on the fungalmembrane in a lung infection model. GlcCer is critical in maintainingfungal cell membrane integrity and represents an attractive therapeutictarget. In addition, it is well-established that gene deletion ofglucosylceramide synthase (Gcs1) results in the creation of a C.neoformans strain, Δgcs1, that does not cause morbidity or mortality ina mouse model of CM. Moreover, Δgcs1 fungi exhibit deficient growth invitro at a pH of >7, a similar pH to that found in the extracellularalveolar space in the lung where Cn thrives 10 and is the predominantfirst site of infection.

There is a major clinical need for new drugs due to a dramatic increaseof morbidity and mortality by invasive fungal infections. Without beinglimited by a particular theory, the compounds contained herein decreasethe synthesis of fungal but not mammalian GlcCer. This action seems tobe specific to the transport of fungal ceramide species. The compoundsare active in vitro against fungi, especially C. neoformans, P. murina,P. jiroveci, R. oryzae, and dimorphic fungi. The compounds appear to beeffective in vivo against cryptococcosis, candidiasis and also againstpneumocystosis. The compounds do not induce resistance in vitro and theyare synergistic with existing antifungals.

C. albicans is resistant in vitro but not in vivo. Studies performed inthis fungus have suggested that GlcCer is important for virulence butthrough a mechanism other than facilitating growth at neutral/alkalinepH, which is the pH used to screen our ChemBridge library. Hence,inhibition of GlcCer in C. albicans does not block fungal growth invitro. However, because the compound still decreases GlcCer synthesis,which is required for Candida virulence, the treatment is effective inpartially protecting mice from invasive candidiasis. These findingssupport previous studies suggesting that the effect of GlcCer in vivoduring Candida infection goes beyond the regulation of fungal alkalinetolerance.

The compounds disclosed herein inhibit GlcCer synthesis; however, thislipid is most likely not the only target of these compounds. In fact,the blockage of fungal growth in alkaline pH due to the loss of GlcCer(Δgcs1 mutant) can be restored if Δgcs1 cells are shifted to an acidicenvironment (Singh A. et al. 2012). This can occur even after the cellsare left in cell cycle arrest for 72 hours. This means that the lack ofGlcCer has a “static” effect on cell growth. However, the compoundsdisclosed herein kill fungal cells. One explanation for this effect isthat treatment with the compound acutely leads to the accumulation ofsphingosines, which is highly toxic to fungal cells (Chung, N. et al.2001; Chung, N. et al. 2000). The accumulation of sphingosine species isnot present when Gcs1 is deleted (Rittershaus, P. C. 2006) or inmammalian cells treated with compound. Thus, the effect seems to gobeyond the inhibition of GlcCer and this may account for the fungalkilling effect exerted by the compounds and not by the absence ofGlcCer.

In summary, molecules were identified that target the synthesis offungal but not mammalian GlcCer. These hydrazycins have potentantifungal activity in vitro and in vivo against a variety of clinicallyimportant fungi. They also displayed synergistic action with currentantifungals, low toxicity, favorable PK parameters, and fungal specificmechanisms of action.

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What is claimed is:
 1. A compound having the structure:

wherein R₁ is —H, alkyl, alkenyl, or alkynyl; R₂ is —H, alkyl, alkenyl,or alkynyl; R₆ is —H, halogen, C₁-C₆ alkyl, —OH, —CHF₂, —CF₃, —OCHF₂ or—OCF₃; R₃, R₄, and R₅ are each independently —H, halogen, C₁-C₆ alkyl,—OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, or R₃ and R₄ areeach independently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl),—CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆ combine to form a fused arylor fused heteroaryl, which are each unsubstituted or substituted, or R₃and R₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅ combine to form afused aryl or fused heteroaryl, which are each unsubstituted orsubstitute, or R₅ and R₆ are each independently —H, halogen, C₁-C₆alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₃ andR₄ combine to form a fused aryl or fused heteroaryl, which are eachunsubstituted or substituted; and A is a substituted monocyclic aryl ora substituted monocyclic heteroaryl, wherein the substitution is withhalogen, alkynyl, alkoxy, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂, —OCF₃, —CN, —CH₂OCH₃, —N(CH₃)₂, —CH₂F, —N₃ or —CCH, or anunsubstituted or substituted bicyclic aryl or bicyclic heteroaryl, or

wherein when R₃, R₄ and R₆ are each —H and R₅ is —OH or —OCH₃, or R₃, R₅and R₆ are each —H and R₄ is —Br, then A is other than ortho-tolyl ormeta-bromophenyl, or a pharmaceutically acceptable salt or esterthereof.
 2. The compound of claim 1 having the structure:

or a pharmaceutically acceptable salt or ester thereof.
 3. A method ofinhibiting the growth of a fungus, or inhibiting fungal sphingolipidsynthesis in a fungus, or inhibiting fungal sphingolipid synthesis in afungus in a mammal without substantially inhibiting mammaliansphingolipid synthesis, comprising contacting the fungus with aneffective amount of the compound of claim 1 or a pharmaceuticallyacceptable salt or ester thereof, so as to thereby inhibit the growth ofthe fungus, or inhibit fungal sphingolipid synthesis in a fungus, orinhibit fungal sphingolipid synthesis in a fungus in a mammal withoutsubstantially inhibiting mammalian sphingolipid synthesis.
 4. The methodof claim 3, wherein the compound has the structure:

or a pharmaceutically acceptable salt thereof.
 5. The method of claim 3,wherein the amount of the compound and the amount of the anti-fungalagent when taken together is more effective to inhibit the growth of thefungus than the anti-fungal agent alone, more effective to inhibitfungal sphingolipid synthesis than the anti-fungal agent alone, or moreeffective to inhibit fungal sphingolipid synthesis without substantiallyinhibiting mammalian sphingolipid synthesis in the mammal than theanti-fungal agent alone.
 6. A method of inhibiting the growth of orkilling a fungus in a subject or treating a subject afflicted with afungal infection comprising administering to the subject an effectiveamount of the compound of claim 1, or a pharmaceutically acceptable saltor ester thereof, so as to thereby inhibiting the growth of or kill thefungus in the subject or treat the subject afflicted with the fungalinfection.
 7. The compound of claim 1 having the structure:

wherein R₁ is —H, alkenyl, or alkynyl; R₂ is —H, alkyl, alkenyl, oralkynyl; R₅ is halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃,—OCHF₂ or —OCF₃; R₆ is —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl),—CHF₂, —CF₃, —OCHF₂ or —OCF₃; R₃ and R₄ are each independently —H,halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃, or R₃ and R₄ are each independently —H, halogen, C₁-C₆ alkyl,—OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₅ and R₆combine to form a fused aryl or fused heteroaryl, which are eachunsubstituted or substituted, or R₃ and R₆ are each independently —H,halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃, and R₄ and R₅ combine to form a fused aryl or fused heteroaryl,which are each unsubstituted or substitute, or R₅ and R₆ are eachindependently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃, and R₃ and R₄ combine to form a fused aryl orfused heteroaryl, which are each unsubstituted or substituted; and A isa substituted monocyclic aryl or a substituted monocyclic heteroaryl,wherein the substitution is with alkynyl, alkoxy, —O—(C₁-C₆ alkyl),—CHF₂, —OCHF₂, —OCF₃, —CN, —CH₂OCH₃, —N(CH₃)₂, —CH₂F, —N₃ or —CCH, or anunsubstituted or substituted bicyclic aryl or bicyclic heteroaryl,wherein the substitution is with halogen, alkynyl, alkoxy, C₁-C₆ alkyl,—OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂, —OCF₃, —CN, —CH₂OCH₃,—N(CH₃)₂, —CH₂F, —N₃ or —CCH; or A is

wherein when R₃, R₄ and R₆ are each —H and R₅ is —OH or —OCH₃, then A isother than ortho-tolyl or meta-bromophenyl, or a pharmaceuticallyacceptable salt or ester thereof.
 8. The compound of claim 7, whereinthe monocyclic or bicyclic aryl or heteroaryl is substituted with—O—(C₁-C₆ alkyl), —CHF₂, —OCHF₂ or —OCF₃; or wherein the fused aryl orfused heteroaryl is substituted with halogen, C₁-C₆ alkyl, —OH,—O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; or wherein one of R₃-R₆is other than —H; or wherein two of R₃-R₆ is other than —H.
 9. Thecompound of claim 7, wherein R₃, R₄ and R₆ are each independently —H,halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃; or wherein R₃, R₄ and R₆ are each independently halogen,—O—(C₁-C₆ alkyl), —OCF₃ or —CF₃; or wherein R₃, R₄ and R₆ are eachindependently halogen or —O—(C₁-C₆ alkyl); or wherein R₃, R₄ and R₆ areeach independently —Cl, —Br, —F, —O—(C₁-C₆ alkyl), —OCF₃ or —CF₃; orwherein R₃, R₄ and R₆ are each independently —Cl, —Br or —O—(C₁-C₆alkyl), or wherein R₃ is —H, R₄ is —H, R₅ is —CH₃, Cl or Br, and R₆ is—H.
 10. The compound of claim 7 having the structure:


11. The compound of claim 7, wherein R₃ and R₄ are each independently—H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃, and R₅ and R₆ combine to form a fused aryl or fused heteroaryl,which are each unsubstituted or substituted with halogen, C₁-C₆ alkyl,—OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; or wherein R₃ andR₆ are each independently —H, halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₄ and R₅ combine to form afused aryl or fused heteroaryl, which are each unsubstituted orsubstituted with halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂,—CF₃, —OCHF₂ or —OCF₃; or wherein R₅ is halogen, C₁-C₆ alkyl, —OH,—O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, R₆ is —H, halogen, C₁-C₆alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, and R₃ andR₄ combine to form a fused aryl or fused heteroaryl, which are eachunsubstituted or substituted with halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃; or wherein R₅ is halogen, C₁-C₆alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or —OCF₃, R₆ is —H,halogen, C₁-C₆ alkyl, —OH, —O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂ or—OCF₃, and R₃ and R₄ combine to form a fused unsubstituted phenyl; orwherein the compound has the structure:


12. The compound of claim 7 wherein A is substituted monocyclic aryl ora substituted monocyclic heteroaryl, or an unsubstituted or substitutedbicyclic aryl or bicyclic heteroaryl, wherein the aryl is a phenyl,1-naphthyl or 2-naphthyl, or the heteroaryl is a pyridinyl, pyrrolyl,thienyl, furyl, quinolyl, isoquinolyl, indolyl, benzothienyl orbenzofuryl.
 13. The compound of claim 7, wherein A has the structure:

wherein R₇, R₈, R₉, R₁₀ and R₁₁ are each, independently, —O—(C₁-C₆alkyl), —CHF₂, —OCHF₂, —OCF₃, —CN, —CH₂OCH₃, —N(CH₃)₂, —CH₂F, —N₃ or—CCH; or wherein R₇, R₈, R₉, R₁₀ and R₁₁ are each, independently,—O—(C₁-C₆ alkyl), —CHF₂, —OCHF₂ or —OCF₃; or wherein R₇, R₈, R₉, R₁₀ andR₁₁ are each, independently —O—(C₁-C₆ alkyl).
 14. The compound of claim7, wherein A has the structure:


15. The compound of claim 7, wherein A has the structure:


16. The compound of claim 7 having the structure:

or a pharmaceutically acceptable salt or ester thereof.
 17. The compoundof claim 1 having the structure:

wherein A is a substituted monocyclic aryl or a substituted monocyclicheteroaryl, wherein the substitution is with alkynyl, alkoxy, —CHF₂,—OCHF₂, —CN, —CH₂OCH₃, —N(CH₃)₂, —CH₂F, —N₃ or —CCH, or an unsubstitutedor substituted bicyclic aryl or bicyclic heteroaryl, wherein thesubstitution is with halogen, alkynyl, alkoxy, C₁-C₆ alkyl, —OH,—O—(C₁-C₆ alkyl), —CHF₂, —CF₃, —OCHF₂, —OCF₃, —CN, —CH₂OCH₃, —N(CH₃)₂,—CH₂F, —N₃ or —CCH; or A is

or a pharmaceutically acceptable salt or ester thereof.
 18. Apharmaceutical composition comprising the compound of claim 7 and apharmaceutically acceptable carrier, and optionally an anti-fungalagent.
 19. A method of inhibiting the growth of a fungus, or inhibitingfungal sphingolipid synthesis in a fungus, or inhibiting fungalsphingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian sphingolipid synthesis, comprising contacting thefungus with an effective amount of the compound of claim 7 or apharmaceutically acceptable salt or ester thereof, so as to therebyinhibit the growth of the fungus, or inhibit fungal sphingolipidsynthesis in a fungus, or inhibit fungal sphingolipid synthesis in afungus in a mammal without substantially inhibiting mammaliansphingolipid synthesis.
 20. The method of claim 19, wherein the amountof the compound and the amount of the anti-fungal agent when takentogether is more effective to inhibit the growth of the fungus than theanti-fungal agent alone, more effective to inhibit fungal sphingolipidsynthesis than the anti-fungal agent alone, or more effective to inhibitfungal sphingolipid synthesis without substantially inhibiting mammaliansphingolipid synthesis in the mammal than the anti-fungal agent alone.